TYAITEt- +rySJFUlETIBRARY

THE EFFECTS OF CYCOCEL (CCC) ON TOl',tATO UNDER !'IATER STRESS

b

Solomon Amoabin B.Sc. Agric' Hons (Ghana)

Department of Plant PhYsiologY

!,laite Agriculcural Research InstituteThe UniversiÈY of Adelaide

South Australia

Thesis submitted for the Degree of

Master of Agricultural Science

December, 1983.

TABLE OF CONTENTS

STATBMBNT

ACKNOWLEDGEMENTS

SUI',IMARY

ABBREVIATIONS AND SYI'{BOLS

FIGURES

TABLBS

INTRODUCTION

CCC AND PLANT

Page No_.

iii

iiiv

viix

CFIAPTER Ir.1

r.2

CHAPTER IIII.1TT.2

II.3II.4II.5II .6

r.1.1T.L,2

r.1.3r.1.4

GROWTH

The use of cycocel in general agriculturePhysical, chemical and physiological propertiesof cycocelCCC (and other compounds) as plant growth retardantsEffects of CCC on Plant growth

cif t.citvEffect of CCC on vepetative andreDrodu ctive prowth

CCC and nutrient content and uptakeUptake and metabolism of CCC

Mode of ActionCCC-induced resistance to pests and diseases

PHYSIOLOGY OF PLANT I^/ATER STRESS RESISTANCE

Development of l^/aEer stressIndicators of plant water statusMechanism of water stress resisEanceDrought avoidanceStomatal resPonse to water stressChemical induction of stomatal closureDrought tolerancer.2.7.r OsnoresuTationr.2. 7.2 Ti eTasticitv

I.3.1 CCC (and other retardants) and drought resistance

MATERIALS AND I.,ÍETHODS

PLANT MATERIAL

1

1

1

224

4

6

r.1 .4. 1

r.r .4.2

B

I9

11

I.1.5r.1 .6T.T.7r.1.8

T,2.Lr.2.2I.2.3r.2.4r.2.5r.2.6r.2.7

11

1113T415161920

2022

22

Ir.6. 1

TT.6.2rr .6.3

PLANT GROWTH ENVIRONI"IENT

CUJ,TTVATION OF PLANT

PREPARA T]ON AND APPLICATION OF CCC

METHODS OF STRESS I},IPOSITION

I',IEASUREMENT OF PLA NT WATER STATUS

Leaf water potentialLeaf osmoLic potentialRelative water content

25

25

25

25

26

26

27

272828

II .7 MEASUREMENT OF GROh]TH PARAMETERS

Plant IleightLeaf AreaFresh wej-ghtDry weight

ÏI. B LEAF DIFFUS]VE RESTSTANCE. STOMATAL COUNTS

AND TRANSP]RATI ON

II.8.1 Leaf diffusive resistance

II. B. I .l CaTibration of di usive resistance Poronte terrI. B. 1 .2 Measurenertt of Teaf diffusive resistanceII .8. 2.L StonataT counts

TI.II.II.II.

II.8.3II .9

rr.9. 1

rr.9.2

7.r7,27.37.4

Page No.

28

2B292929

29

29

293033

33

34

34

34

35

36

36

36

36

363739

42

424347

4B

484949

TranspirationCHEMICAL PROCEDURES

Determination of ProlineExtraction and assay of quaternary amlnonium compounds

using the N .1"1. R. technique

II. 10 EXPERIMENTAL DESIGN AND STATISTICAL ANALYSIS

RESULTS AND DISCUSSIONCHAPTER IIIIII. l CCC AND WATER STRESS EFFBCTS ON WATER STATUS AND

OF TOMATO

1.1 IntroductionI.2 Effects of differenL concentrations of CCC and

water sLress on l{ater potential and growth

III.1 .2.I IntroductionIII. 1 .2.2 ResuTtsIrr. 1 .2. 3 Discussion

III.1.3 The effects of 1000 ppm CCC on leaf, stem and

root growth

III.l .3.I IntroductionIII.1 .3,2 ResuTtsrIr. 1 .3. 3 Discussion

III.1.4 The effecrs of 1000 ppm ccc applíed at differentstages of growth and water stress on rrrater potentialand growth

III.1 .4.I IntroductionIII.1.4.2 ResuTtsIII.1 .l+,3 Discussion

TIT.2 A COI',IPAR]SON OF PEG_INDUCBD \^IATER STRESS AND SOIL I^/ATBR

DEPLETION BY WITH-HOLDING

GRO\^ITH

IIIIII

(

TREATI'4ENT

IT,T,2.1 IntroductionIII.2.2 ResultsITT.2.3 Discussion

)¿

525356

\^/ATER) COMBINED \^/ITI{ CCC

III.3 CCC AND hIATER RELATIONS OF TOMATO

III.3.1 IntroductionIII.3.2 Relative water content

III .3 .2.I MethodIII.3 ,2,2 nesults

III.3.3 Osnrotic potential and turgor potentialIII.3 .3.L MethodIII.3.3.2 ResuTtsIII.3.3.3 Discussion

III.4 CCC AND ACCUI'4ULATION OF PROLINB AND OTHER

QUATERNARY AI'IMONIUI.{ COMPOUNDS

I]IIITIII

III.5IIIII]III

III.6DIFFUSIVE RESISTANCE

III.6.1 IntroductionIII.6.2 ResultsIII.6.3 Discussion

II].7 EFFECTS OF CCC AND hIATER STRESS ON TOTAL WATER LOSS

.4.1 InLroduction

.4.2 Resultsl4i3 Discussion

lege l{o.

5B

5Bs9

s9s9

63

636367

70

707T79

BO

BO

8187

B8

888B9I96

969699

to2

106

119

EFFBCT OF CCC ON LEAF DIFFUSIVE RES]STANCE

.5.1 IntroducLion

.5.2 Results

.5.3 DiscussionEFFECTS OF CCC AND WATER STRESS

III.7III.7TIT.7

.1)

.3

IntroductionResultsDiscussion

CHAPTER IV GENERAL DISCUSSION

BIBLIOGRAPHY

APPENDICBS

I

STATEMENT

This thesis has not been previously subrnitted

for a degree at this or any other University,

and is the original work of the writer except

where due reference is made in t.he text.

IN

l_ l-

ACKNO\{LEDGEMENTS

I wish to express my sincere thanks to Professor L.G.

Paleg for his guidance ' constructive criticisms and

encouragenent in the course of the programne. I am

also grateful to Dr. D. Aspinall for acLing as a

second supervisor.

I am also indebted to Dr. C.F. Jenner for his guidance

during the absence of my suPervisors, and also toDr. P.E. Kriedemann for his invaluable advice. I am

also grateful to Dr.T.J. Douglas for his help and

advice during my early days in Australia. lly sincere

thanks also go to Ehe technical staff and all the

other members of uhe Department of Plant Physiology

for their cooperation and help in diverse v/ays.

I arn grateful Lo Carol Arnoabin who helped in typing

and showed a lot of patience and understanding duri-ng

the preparation of this thesis.

Finally, the financial support provided by the

Commonwealth Scholarship and Fellowship Plan and the

extra support from The University of Adelaide are

gratefully acknowledged.

IAI

SUMMAT{Y

CCC (2-chloroethyltrirnethyl ammonium chloride) a synthetic plant grorvth

retardant, has been pleviously founcl to increase the resistance of various

plants to clrought. l{o::k clone on this subject mostly attributes the CCC effectto reduced leaf area and i:rcreased root/shoot ratios due to the retardation

of shoot grorvth though there are a few cases where CCC did not retard shoot

groivth but stil1 induced drought resistance. A few workers have mentioned the

involvement of CCC-induced stonatal closure in the drought resistance of

CCC-t,reated plants. Sone workers have associated the CCC-induced drougl'rt

resistance with netabolic changes of CCC per se, and CCC-mediated netabolic

changes in plants under vlateï stress, without melrtioning how these changes are

related to the CCC-induced d:rought resistance.

This study was conducted to cletermine whether CCC affected the drought

resistance of tomato, which is relatively sensitive to water stress, and, ifso, to explore some of the possible nechanisms underlying the effect. Attention

was directed towards the effects of CCC on the tonato plants under stress

independent of its retardation effect on growth.

At a concentration of 100@prn, CCC retarded the growth of the tomato

plants 6 days after its application as soil drench. CCC reduced height, leafarea, fresh and dry weights of leaves and stem, without any retarclation effecton root growth, resulting in an increased root/shoot ratio. Whether CCC

retarded the growth of the plant or not before inducing water stress, the

CCC-treated plants maintained higher water potential especially within the

first few days after with-holding water, such that they did not wilt as quickly

as the non-CCC-treated plants. Despite this prolonged survival under water

stress due to CCC treatment, growth was not sustained. When PEG was used to

induce stress, CCC did not have any effect on water potential and it could not

reduce the toxic effect of PEG, in the forrn of leaflet margin chlorosis.

Under soil water depletion induced by with-holding water from the plants,

RWC was higher in the CCC-treated plants but osmotic potential did not

decrease as rnuch in the CCC-treated plants as the non-CCC-treated plants.

The relationships between water potential and RWC, osmotic potential and turgor

potential were not altered by CCC which indicated that CCC did not enhance the

treated plantst ability to adjust osrnotically. This was supporte<l by the

apparent lack of effect of CCC tTeatment in pronoting solute accumulation.

In normal well-watered plants, CCC caused a rapid differential increase

in adaxial leaf diffusive resistance but not in abaxial resistance indicating

a CCC-induced closure of the adaxial stomata, independent of its effects on growth.

AV

This was consistent rvith a marked decrease in transpirational water loss

from the adaxial leaf surface of the CCC-treated plants. Water stress

per se (induced by wi-th-holding water fron the plants) also caused stomatal

closure but this was quicker in the non-CCC-treated plants than in the

CCC-treated plants. The same hrater potential threshold was found forstonatal closure and effective control of further water loss under stTess,

and this was unaffected by CCC. This indicated that, because of the

initial CCC-induced adaxial stomatal closure and the concommitànt reduced

transpiration, the water potential of the CCC-treated plants declined less

rapidly as the stress progressed, and that the time required to reach the

water potential threshold for stomatal closure and effective control ofI^Jater loss was prolonged by the CCC treatment.

It was concluded that, independent of its growth retardation effect,CCC enabled plants to delay the onset of severe internal water deficit and,

therefore, prolonged survival through CCC-induced adaxial stonatal closure

and the attendant decreased transpirational water loss. This did not seem

to involve any CCC-induced osrnotic adjustnent. When CCC retarded growth,

however, the increased root/shoot ratio and the reduced leaf area could be

additional factors contributing to the CCC-induced resistance to water

stress.

2

ABA

ATP

14c

CaOC

CCC

cm2

cm

c0 zD^0

Z:dn?

F.C.

Fig.g or-gm

IH,

HC1

H^0¿

hr(s)i. e.

ins.K

J-K.

KCl

kg

IMe0H

Mg

M.!'1.

I'fl,JC_, -1pE.m "s -

ug

¡r1

mg

ml

mm

v

ABBREVIATI ONS AND SYIVIBOLS

abscicic acid

adenosine triPhosPhate

radioactive carbon

calciundegrees centiglîade

cycocelcentimetre ( s )

square cent-imetre(s)

carbon dioxide

deuterium oxide

square decimetre(s)

field caPacitY

figuregram( s )

hydrogen ion

hydrochloric acid

water

hours

that isinchespotassium

potassiurn ionpotassium chloridekilogram(s)litre (s )

methanol

magnesium

molecular weight

r:nethanol : chloroform : water

microeinstein per square meLre per second

microgram(s )

microlitre ( s )

milligram(s)rnillilitre ( s )

millimetre (s)

vl_

NZ niLrogen

N normal

NMR nuclear magneLic resonance

No. number

P phosphorus32P radioactive phosphorus

PEG polyethylene glycolppm parts Per mÍ-llion86nu radioactíve rubidium

rpm revolutions Per minute

R\,\lC relative !ùater contenL

Sec. second(s)

Si siliconet time

t-buOH (tert-Bu0H) tertiary butanol

^t change in time

7" percent

tl h¡ater potential

Us osmotic (solute) Potentialüp turgor potential

{rm matric potential

Fig. I. 1

Fig. I.2

Fig. II.1

Fie. III.1

Fig. IIl.2

Fig. III.1.3

Fig. III.1.4

Fig. III.1.5

Fig. III. 2. 1

Fig. III.3.1

Fig. III.3.2

Fig. III.3.3

Fie. III.3.4

vl_r

FIGURES

Structural representation of some plant grorvthretardants (Cathey, 1964).

Metabolic scheme for the mode of action of CCC

by Stoddart (1964).

Relationship between the time lapse (Ai) anddiffusive resistance in the calibration of theporometer at 20"C and 24"C.

Effects of different levels of CCC preEreatmenton waLer potential during 4 days of with-holding water.

Photographs showing CCC-treated and control plantsunder well-watered condition and under stressinduced by with-holding 'iúater. The CCC-treatedplants were supplied with 1000 ppm CCC.

Retardation effects of 1000 ppm CCC and waterstress.

Effect of 1000 ppm CCC on (a) leaf area and (b)leaf length, with respect to leaf position,5 days and 16 days after t.he CCC treatment.

Effects of 1000 ppm CCC supplied at differentstages of growth and water stress on waterpotential.

Effect of CCC on water potential under PEG-inducedstress and soil moisLure deplet.ion induced bywith-holding water.

Effect of CCC and water stress on water potential.

Effects of CCC and water stress on RtrtlC.

Relationship between Rl'/C and waLer potent.ial(moisture release curve).

Effect of CCC on water potential under sËressand recovery.

Effect of CCC on osmotic poEenLial under stressand recovery.

PaRe No.

I2

32

55

60

61

5

3B

40

44

46

50

62

64

65Fig. III.3.5

vlal-

Relationship between water potential and

osmotic potential.

Effect of CCC on turgor potential under stressand recovery.

Relationship between water potential and turgorpotential.

Effect of CCC and water stress on water poLential.

Effects of CCC and water stress on prolineaccumulation.

Effects of CCC and water stress on h¡ater Potential.

stress on prolineEffects of CCC and wateraccumulation.

NMR spectrum of an extract of a CCC-treatedplantl s leaf, showitrg choline, CCC and prolinepeaks.

NMR spectrum of an extract of a non-CCC-treatedplantl s leaf, showing choline and proline peaks '

Diurnal pattern of (a) adaxial and (b) abaxialdiffusive resistances in CCC-treated and controlplants, in the glasshouse.

Diurnal pat-tern of (a) adaxial and (b) abaxialdiffusive resistances in CCC-treated and controlplants, in the growth room.

Effect of CCC on adaxial diffusive resistancewithin a period of 7 daYs.

Effect of CCC on adaxial diffusive resistanceunder stress and recoverY.

Effect of CCC on abaxial diffusive resistanceunder stress and recoverY.

Relationship between water potential and adaxíaldiffusive resistance.

Relationship between v/ater potential and abaxialdiffusive resistance.

Relationship between vraLer poEential and adaxialconductance.

lqge No.

,'IÈl!r:

Fig. III.3.6

Fig . III.3.7

Fie. III.3.B

66

6B

69

12

73

74

75

76

77

82

B3

B4

89

90

92

93

94

Fig.

Fig.

III.4. 1

TTT.4.2

Fig. III.4.3

Fie. III.4.4

Fie. III.4.5

Fig. III.4.6

Fig.III.5.1

Fig. III.5.2

Fig. III.5.3

Fig. III.6.1

Fig. II'I.6.2

Fig. III.6.3

Fig. III.6.4

r

Fig.lII.6.5

Lx

I

,l

I'i

I

'l.lI

l

l

I

I

I

Fig. III.6.6

Fig. III.7.1

Fig. IrI.7 .2

Fig. III.7.3

Fig. TII.7.4

Fig. l

Fig"2

Fig.3

Relationship beLween water potential and abaxialconductance.

Effects of-CCC and water stress on !'Iater loss/day'

Effects of CCC and water stress on cumulativewater 1oss.

Relationship between hraÈer potenLial and waterloss/day.

Relationship between water potenti-al and

cumulative water 1oss.

APPENDIX FIGURES

Photograph showíng the method of determinationof tránspiration rate in excised leaves asdescribed in Section I1.8.3

Photographs showing sLomatal distribution on

the adaxial surface.

Photographs showing stomatal distribution on

the abaxial surface.

Page No.

95

97

100

101

119

722

L23

9B

fË

r":

ii

í

I

(î'

T'I

l

I

x

TABLES

Physical and Chemical Properties of CCC.

Calibration values of che diffusive resistanceporometer at 20"C and 24oC.

Effects of different levels of CCC and rvaterstress on growth characteristics after 4 daysof with-holding water.

The effects of 1000 ppm CCC on various grok¡thcharacteristics of well-watered tomato plants '

Effects of 1000 ppm CCC applied at different times(Early and LaLe) on growth characteristics beforeand after stress.

EffecLs of CCC on various gror'rth characteristicsbefore the imposition of stress.

Effects of CCC and PEG-induced stress orl variousgrowth characteristics.

Proline, choline and CCC content of leafesLlmated by the NMR technique.

Relationship between plant si-ze, diffusivereistance and transPiration.

Diffusive resistance of intact plant and trans-piration rate of the Adaxial and Abaxial surfacesóf excised leaves. Measurements were taken5 days after CCC treatment.

APPENDIX TABLB

Number of stomaLa/rnm2

Table I.1

Table II.1

Table III. l

Table I]J.2

Table III.3

Table III.2.1

Table III.2.2

Table III.4.1

Table III.5.1

Table III.5.2

Table 1

31

4L

45

51

57

78

86

r2r

5l+

85

I

r

1 \¡//rl'i I lt lS'íIIUTE

LIBRAITY

CI]APTER I

INTRODUCTION

r.1 CCC AND PLANT GROWTII

I.1.1 The use of cycocel in general agriculture

The ultimate goal of Plant Breeders is to incorporate as many

desirable characteristics as possible into a given plant. while this feat

is really difficult- to achieve, some of the desired characteristics can be

produced chemically, through the application of plant growth regulators.

Cycocel (2-chloroethyltrimethyl ammoniurn chloride) is a

synthetic plant growth regulator and , more specifically, a plant grolvth re-

tardant. So far, knowledge available indicates that it is used commercially

on ornamental crops such as poinsettias and azaleas to produce desirable

plants, on cereals especially wheat to prevent lodging in many cases and

increase yield as well in some cases and on some varieties of grape to enhance

fruit set.

Fromtheliterature,therearePotentialusesofcycocelonanumber of fruit, vegetable, field and other miscellaneous croPs which include

the following:

(i) Ultimate increase in Yield(ii) ComPact Plants(iii) Promotion of flowering(iv) Resistance to certain insects and plant diseases

These potential uses of cycocel will be outlined in the literature '

Furthermore, owing to the versatility of the chemical in the

sense of species and varietal responsiveness, tirne of application, dosage,

method of application ancl interaction with environmental conditions, it opens

a broad spectrum for research work. Though quite a substantial amount of

work has already been done, there is stil1 the need for a lot more to properly

explain some of the morphological, physiological, anatomical and biochemical

changes in plants attributed to the chemical and possibly open ner'{ areas of

usage.

2

T.T.2 Physical, chernical ancl physiologicalproperties of cycocel

The physical and chemical propertie,s of CCC are presented inTable I.1. However, attention must be draln t-o the naming of the compound.

It has a trade name of Cycocel and a generic nante of chlornequati, the chemical

names being 2-chloroethyltrimethyl ammonium chloride or chlorocohline chloride

- commonly abbreviated as CCC. For convenience, the chemical will be referredto as CCC throughout this thesis.

The first observable effecl due to CCC treatment on plants isintense greening of leaves. This precedes the shorlening of sterns and overallreduction in plant size as reported by Tolbert (1960b). General responses ofplants as a result of treatment wit.h CCC include the following:

(i) Sturdier and nore compact plants (l'littwer and Tolbert, 1960;

Tolbert, 1960b).

(ii) Shortened and thickened internodes and in some plants general

reduction in plant stature (Cathey, 1964; Tolbert, 1960b).

(iii) Intense green leaves (I{ittwer and Tolbert, 1960; Tolbert, 1960b).

(iv) Lodging resistance and increase in yield in some cereals grains

(Humphries , 1968 )

(u) Resistance to envj-ronmental sLresses, e.g. drought and salinity(Halevy and Kessler, 1963; El Damaty et a7., L964)

(vi) Promotion of flower bud initiation and development (Cathey, 1964)

(vii) Resistance to certain insects and diseases (Tahori et a7., I965a

& 1965b).

r.1.3 CCC (and other compounds) as plant growth retardants

CCC fa1ls within the category of chemicals designated"PTant Growth

Retardants" (Cathey, L964; Tolbert, 1960a). These are synthetic organic

compounds which do not occur naturally in plants but when applied exogenously

slow celI division and elongation in the shoot tissues, and regulate plant

height physiologically, and indirectly affect flowering without malformations

of the plants (Cathey , 1964). Thus growth inhibitors (e.g. maleic hydrazide)

auxins, herbicides and gerrnination inhibitors which ultimately stunt or

completely suppress the growth of plants andfor cause visj-b1e malformations

of the leaves, stems and flowers of the treated plants are excluded from the

growth retardant group.

3

TABLE I.1: Physical and Chemical Properties of CCC

Characteristic

Chemical names

Generic name

Empirical formula

Molecular weight

Solubility(i) Water(ii) Methanol(iii) Ether and other hydrocarbons

Hygroscopicity

Melting point

0dor

Physical form

Persistence in soil

Time to noticeable resPonse aftertreatment,

Spray applicaLion

Soil application

Plant spectrum

Toxicity

Bffect

(i) 2-chloroethyltrimethylammonium chloride

(ii) Chlorocholine chloride (CCC)

Chlormequat

158. 1

CompleteCompleLeInsoluble

High

245"C

Fish-like (typically amine)

t'lhite crystalline solid

3-4 weeks

1 week or less

Relatively effective

Effective

Many plants

Yellowing around veins at highconcentration

c5H13c12N

4

The chemicals referred to as grorvth retardants can be grouped

under different families with specific structural requirements for activity.As summerized by Cathey (1964), these include:

(i) Nicotini.urxs - e.g.2,4-DNC (Fig. I.1, compound 1) rvhich

requi-re chloride substitution for activity (M:-tche11 et a7. ¡

1949)

(ii) Quaternary annonium carbanat-es - e. g. AM0-1618 (Fj.g. I.1,compound 2) in which the reduction of any of the basic

moieties, namely the carbamate nitrogen ' the terpene ring, the

quaternary nitrogen.and the halide salt, removes activity(Wirwi1le and l"litchell, 1950)

(iii) Hydrazines - e.g. BOH (Fig. I.1' compound 3), in which the only

requirement for activity is the C-C-N-N chain (Gowing and Leeper,

195s)

(i") Phosphoniums - e.g. Phosphon D and Phosphon S (Fig. I.1, compounds

4a and 4b); the tributyl quaternary phosphonium cation of thisgroup is indispensable for activity and the benzene ring requires

a small nucleophilic and non-ionizable substituent in the

4-position (Pretson and Link, 1958).

(u) Substituted cholines - e.g. CCC (Fig. I.l, compound 5), the

trimethyl quaternary ammonium cation is necessary for activity inthis group. These compounds are analogs of choline with the

general formula CH2X-CH2-N-(CH3)3 and for activity, a sma1tr nucleo-

philic and non-ionizable substituent at X is required. CCC is the

, chloride sa1t, however the bromide salt is also active (Tolbert,

1960a).

(vi) Substituted naTeanic and succinamic.acid - e.g. C011 and 8995

(Fig. I.1, compound 6). This group differs from the others inthat it does not contain a benzene ring, quaternary ammonium or

phosphonium cation or small nucleophilic and non-ionizable substi-tuents (Cathey , 1964).

I .r.4 Bffects of CCC on plant growth

I. 1 .4.1 Specificitv

Responsiveness of plants to plant growth retardants is unrelated

to taxonomic classification. Species vary in their response to various

growth retardants (Cathey and Stuart, 1961), and even different cultivarsof the same species vary in their responsiveness to the applied chemical

(Cathey, 1964). As compared to AM0-1618 and phosphon, many plant species

5

FIG. I.1: StrucLural representation of some p1anl growth

retardants (CatheY , 1964).

1) 2,4 DNC

2) Ano-1618

lI-(2, 4-d ichlorobenzyl ) -1 -methyl -2-3-pyridyl, pyrrolidinium chloride]

[ 4-Hydroxyl-5-isopropyl-2-methylphenylLrimethylamnonium chloride, l-pi-peridine carboxylate]

[ $-hydroxye thylhyd r azínel

[ 2, 4-dichlorobenzyl-tributylphosphoniumchloride l

[ 2, 4-dichlorobenzyl-tributylammoniumchloride l

[ (2-chloroethyl) trimethylammonium chlori-de]

IN-dimethylamino maleamic acid]

3)

4a)

4b) Phosphon-S

BOH

Phosphon-D

ccc

8995-(B-nine )

s)

6)

cl-H¡1 Cl-CHz-C

5

H.'Cl-

clHs

'CHt-¡H-\' YH"-6ooH

3

.crH¡2

3HO-CHzCH HNHz

Hç

C.Hc'Cl4a

Hç

cl c 'Cl-4b

6

are responsive to ccc. out of 55 spccies tested bycathey ancl St'uart

(196r), only 6 were responsive to A110-1618, 19 r:esponsive to phosphon and

44 responsive to ccc. cathey (1975) r:eported that out of BB species

tested,5 responcled to Aì40-1618, 12 responded to phosphon and 21 responded

to CCC. One Ehing peculiar with these growth retardants is that though

CCC is effective on a broader spectrum of plants it does not necessarily

follow that a plant responsive to AM0-1618 and/or phosphon will be re-

sponsive to CCC and vice versa.

Furthórmore, the method of application of the retardant affecLs

the responsiveness. ccc applied to dahlia as foliar spray was less

effective than as soil drench (Bhattacharjee et a7., 1971)' 0n rvheat'

soil application of ccc \^ras more effective on light soil but on clay only

spraying shorLened the straw (Humphries, 1968)"

Growth retardants are generally more effective on dicotyledoneous

plants than- monocotyledoneous plants (Cathey , 1964) ' Among cereal

crops wheat has been very responsive especially to CCC; however, there

is differential sensitiviLy among the varieties that are retarded by CCC'

r.r .4.2 Effect of CCC on vesetative and reprodu ctive Erowth

As mentioned earlier the first ' most easily observable response

to CCC (as well as to other growth retardants) is intense greening of leaves

and this is either attended with or followed by shortenì-ng and thickening of

stems. Retardation of stem growth by CCC rnight be attrjbuted to an inhi-

bition of cel1 division in the subapical meristem as reported by Sachs et a7 '

(f960). In wheat, Russel and Kimmins (1972) observed that CCC treatment

decreased the total number of cel1s but the number of cells per unit fresh

weight and per unit dry weight increased which suggested a decrease in cell

size. They inferred, therefore, that CCC inhibited meristematic activity

and cell elongation. Retardation of stem growth by CCC may be accompanied

by an increased transverse growth. Sachs and Kofranek (1963) observed

that CCC stimulated stem transverse growth in Chrysanthenun and this lateral

expansion was attributed to the enlargement of cells ' Zeevarl (1965)

reported that the diameter of pith pArenchyma cells in Pharbitis seedlings

were larger in ccc-treated plants than in untreated plants.

The effect of ccc on leaf growth is variable. A common visible

effect is the intense green colour of leaves of CCC-treated plants ' CCC

reduced leaf area of sugar beet (Humphries and French, 1965), and tomato

(Pisarczylc and Splittstoesser , IgTg)' Dyson (f965) showed that CCC

7

decreased the leaf area of potato and the higher the c'or-icentration of CCC

the more the reduction in leaf area. ccc applied Eo Phaseo'Lus vulgaris

resulted in a reduction in leaf area which nost probably rvas due to t'he

reductio. i. the rate of expansion of leaves (Felippe and Dale ' 1968).

0n Ehe contt'ary, mustarcl plants treated with CCC responded by an increase

in total leaf area which resulted frorn production of more lateral leaves or

enlargement of stem leaves (Humphries, 1963). CCC did not have any signi-

ficant effect on leaf lamina area in wheat (Humphries et a7.¡ 1965)'

several reports indicate that ccc either retards root gro\^/th less

tha' shoot growth or stimulates root growth. In wheat, cCC enlarges the

root system to an extenc which depends on the variety ' as rePorted by

Humphries (1968). According to llumphries, ccc increased root weight' area

of absorbing surface and the amount of roots at all soil depths when applied

to wheat. As usual with ccc, this proposecl stimulation of root growth does

notapplytoallspecies.Notsurprisingly'therefore'CCCretardedrootgrowth at higher concentrations when applied to Norway Spruce (Dunberg and

Eliason, 1972).

Generally,cccretardsthegrowthofvariousplants;nonetheless,this effect is not universal. In a few cases ccc, especially at low con-

centrations, stimulates growth, for example, in peas as reported by Adedipe

et aL (1968). In snapdragon, llalevy and \^/ittwer (1965a) reported that a

foliar spray with ccc increased stem height and dry weight of leaves and

stem but soil application had no effect. \{ünsche (1969) found that while

a foliar spray of ccc stimulated sLem elongation in snapdragon, soil

application retarded growth even at lolu concentrations ' However ' in

gladiolus, ccc applied as soil drench stirnulated growth (Halevy and shilo '

1970). ccc srimulated growth in lemons (llonselise eË al" 1966) and

begonia (l.leicJe, 1969), and, in tomato, an initial growth retardation due

to CCC was followed by growth promotion (Van Bra8t' 1969)'

Apptication of ccc in certain cases caused suppression of vege-

tative growth and prompted flower initiation (Cathey, 1964) ' hlittwer and

Tolbert (1960) reportecl that CCC applied to the roots of tomato promoted

earlier flowering. Abdul et a7. (1978) observed that ccc increased the

nurnber of florvers formed in the first inflorescence and also reduced flower

abortion in tomato plants grown at high temperature with low 1i ght '

Abdalla and verkerk (1970) observed a reduction in flower drop and in-

creased fruit-set and development in tomato plants treated with CCC through

soil application. ccc drenches or sprays induced flower buds to form

B

earlier on azaleas than on untreatecl plant-s (Cathey and Stuart,196f).

Junt.illa (l9BO) reported that CCC Íncluced flower bud formation on immature

plants of Sa-lix pentandta as well as on cuttings from seedl-ings' Coombe

(1965) showed that in some \¡arieties of grapes, ccc increased the fruit

set and cluster weight. In sone species ' for example in Pharbitis ni7 'CCC inhibits flowering as reported by Zeevart (1964) '

I.1.5 CCC and nutrient content and uptake

Evidence available suggests that ccc treatment results in an

increased concentration of macronutrients (N, P, Ca and Mg) but not K which

is reduced (Robinson, Ig75). Knavel (1969) 31so reported that tornato plants

treated t/ith CCC as foliar spray contained more N' P' Ca and Mg but less K

than untreated plants. In a similar nìanner' young Poinsettia plarrts treated

wirh ccc had more N and P but lacked K and si (crittendon and Kiplinger' 1969)

and in pea plants, CCC treatment increased N, P and llg in the vine but

decreased K (AdediPe et a7.,1969).

El-Fouly et a7. (1970) reported that CCC inhibited totat 32p uPtake

in cotton seedlings, and both foliar spray and addition to nutrient culLure of

CCC resulted in accumulation of 32P in stem but decreased levels in the root '

However, foliar spray increased, while adclition of CCC to nutrient mediun

decreased, the 32P.ont"nt in the leaves. Contrary, Gholke and TolberL (1962)

found that the addition of CCC to the nutrient medium of barley seedlings

resulted in a 3-4 fold increase in total 32P uptuk.. This might have arisen

from the several-fold increase in 32P ,.r the roots, bul- 32P .orrt"nt in the

leaves of CCC-treated plants rvas less than in untreated plants ' Adedipe and

ormod (Lg72) reporred that at high P rate, ccc did not have any effect on the

total P uptake or on relative distribution in the leaf, stem and root; but at

low P rate the root, rel-ative to the leaves and stem, retained more P at

lOOmg/l CCC. Halevy and \,,iittwer (1965þ)shovred that CCC applied to the solution

culture of bean plants resulted in a reduced mobil ity of 86Rb ao the upper stem

but increased it to the roots; however, there l'/as no effect on initial uptake

,ohun B6Rb was applied to one of the expanded primary leaves. It stands to

reason, therefore, Lhat CCC affects the uptake, translocation and distribution

of nutrj-ents in different species differently though there is the general

tenclencyfor ccc to reduce tlie uptake of some nutrients.

Uptake and metabolism of CCC

It has been sqggested that ccc could be taken up by plants eitherr.1.6

9

through the leaves or roots (Dekhuijzen and Vonk, L974; Lord and hrheeler,

1981; Birecka , 1967 ) rhough Blinn (1967) r:eportecl that it is absorbed slowly

from folj-ar deposits on wheat. l4c-rabelled CCC appliecl to the leaf showed

greater movenent to the leaf tip, younger leaves, main stem and ear but less

to t¡e root in wheat t-han in barley and there was greater loss of labelled CCC

in wheat than in barleY.

According ro Blinn (Lg67¡ 14C-f.ue11ed CCC in wheat v/as not meta-

bol-ized in thaL the label-led CCC was the only radioactive-labelled substance

found in the wheat foliage, roots and grain. In support of this, Birecka

(196l) found that CCC \^/as not ¡netabolized in rvheat plants and that the increased

amount of choline he observed in his work did not show any label from the 14C-

labelled CCC. This led him to infer that CCC might have blocked some enzynes

involved in the metabolism of choline. 0n the other hand, Schneider (1967)

showed that labelled choline and other 1abe1led unknown metabol.ites resulted

from labelled CCC-treated barley and Chrysanthemun shooLs. Dekhuijzen and

Vonk (1974) found that CCC was converted to choline which was further meta-

bolized to betaine which, upon demethylation yields finally glycì-ne and serine,

they proposed that serine might have been formed from glycine with the evolution

of. l4COr, during photorespiraÈj-on. The degradation of CCC in wheat to choline

is also supported by evidence from E1-Fou1y and Jung (1969).

I.1.7 Mode of Action

Because CCC affects different species ancl even different cultivars

of Uhe sane species differently, it has been difficult to propose a single

well-defined mode of action for the chemical. Though it is quite logical for

one to argue that CCC may act differently in different plants and under

different conditions, the most popularly held view on the mode of action of

the retardant involves its ínteraction with gibberellin.

Tolbert (1960Þ) and \nlittwer and Tolbert (1960) observed that the

effects of CCC on plants l¡/ere opposite to those of gibberelh-n and these effects

of CCC were reversed by gibberellin. It has also been found that CCC treatment

resulted in the decrease of endogenous gibberellin in some plants (Jones and

Phillips, Lg67 Zeevar:,1966). Paleg et a7. (1965) repor:ted that the inter-

ference of CCC with the gibberellin might arise in five general h¡ays, viz:

(i) Inhibirion of the biosynthesis of endogenous gibberellin(ii) Decrease in the level of compound or class of compounds on or

v¡ith which gibberellin acts or reacts

(iii) DestrucLion or inactivation of gi-bberellin

(iv) An action preventing gibberellin from fulfilling itts primary

or hormonal role

10

(u) Blocking of the physiological response of plants to gibberellins

The above authors favoured the first possibì lity - the inhibition of gibber-

ellin biosynthesis - as the most suitable mode of action of CCC. Several

studies lend supporL to the CCC inhibition of gibberellin biosynthesis.

Lockhart (1962) reported that CCC partially blocks the systeu providing

active gibberellins in bean (PhaseoTus vulgaris) plants. Kende et a7. (1963)

reported that CCC completely inhibited formation of gibberellins in Fusariun

noniliforr¡re and this rvas extended by Ninnernann et af. (1964) who found that

10 ppm of CCC completely suppressed gibberellin production by the fungus and

that the destruction of gibberellin by CCC rvas not observed. Further

evidence came from Harada and Lang (1965), Cross and luleyers (f969) and Barnes

et a7. (1969) rvho located the CCC-inhibited steps in the gibberellin bio-

synthetic pathway.

Although the CCC-inhibition of gibberellin biosynthesis has been

the most frequently reported mode of action of the chemical, there is evidence

to show that it is not the only possible mechanism. In tomato planLs, Van Bragt

(1969) showed that CCC-treated plants conl-ained slightly more gibberellin-likesubstances compared with control plants and, therefore, suggested that CCC did

nor inhibit gibberellin synthesis. Similarly, Halevy and Shilo (1970)

reported of an increase in endogenous gibberellin in gladioli plants treated

wirh CCC. Reid and Crozíer (1970) observed, in pea seedlings, that CCC

treatrnent resulEed in an increase in gibberellin without there being any

para1lel stimulation of growth, which indicaLed that, in peas, the predominant

factor in CCC-induced inhibition of stem growth may not be related to an

effecr of CCC on gibberellin biosynt.hesis. Devay et a7. (1970) were of the

opinion that, in PhaseoTus vuTgaris, the inhibition of the synthesis of

giberellin-like substances hras not the source of the physiological effect of

CCC but, rather, the source could be an acLivation of a system which regulates

the phytokinin level.

There are other modes by which the effects of CCC could be accountecl

for aside from the involvernent of hormones. Heatherbell et a7. (1966)

suggested that in etiolated pea seedlings, CCC might act as an uncoupling agent,

thus resulting in a block of the flow of energy into growth processes due to

reduced mitrochondrial ATP producLion. Adedipe and Ormrod (1970) showed a

decreased ATP level in pea tissue at low CCC concentration suggesting phosphate

utilization as a regulatory site in the growth retarding effect of CCC.

Wittwer and Tolbert (1960) noted that the structural sjmilarityof CCC to choline suggested a possible function in lipid metabolism and the

11

specificity of nel-hyl. groups in the ammoníum cation implied that 14C-1abe11ed

CCC was partially incorporated into phosphatidyl ctioline and lysophosphatidyl-

choline. Douglas (f974) found that CCC jnhibited sterol biosyntiresis in

tobacco seedling at the squalene -2,3-epixode cy6lsse step.

Stoddart (1964) reporLed that CCC uncoupled assimilation and

growth ín Loiiun tenuTentuLl plants. His evidence confirmed that there lvas a

preferential conversion of carbohydrate to amino acids while nitrogen was

adequate but fructosan was forned while nitrogen was inadequate. He therefore

inferred that polymerization of free sugars, which should have been util ized

to sustain growth, to storage polysaccharirles occurred jn the Presence of CCC.

Froni his work, he visual.ized a metabolic scheme (Fig. I.2) showing sites at

which CCC rnight block plant metabolism.

r.1.8 CCC-inducecl resistance to pests and diseases

In some plants, CCC treatment results in their resjstance to sone

insect pests and diseases. Tahori et a7. (1965a) reported that cotton leaf-,

worm larvae did not cause as much destruction on bean plants sprayed lvith CCC

as compared to the untreated plants. The same authors (1965) found that

sLem bases of oleander held in CCC solution were less infested with aphids as

compared with the control. Van Emden (L964) observed that the populatin of

cabbage aphid on brussel sprout was reduced on the plants treated with CCC as

a soil drench.

CCC showed significant control of bacterial spot due to

Xanthononas vesicatoria on pepper plants (Crossan and Fieldhouse , 1964) '

Rarvlins (1962) reporred that CCC inhibited the multiplication of tobacco mosaic

virus (Tl',lV). According to Sinha and ülood (L964) tomato plants treated with

CCC showed a decreased level of verticillium wilt infection' Bean seedlings

treated with CCC and infecÈed with stem rot fungus (ScTerotiun roffsü ) were

less diseased than the untreated plants (Tahori et a7., 1965b). Diercks

(1965) reported that treatment of winEer wheat with CCC preventecl lodging'and

reduced the incidence of stalks infected with eye-spot disease '

T.2 PHYSIOLOGY 0F PLANT I^IATER STRES S RES]STANCE

I.2.I DeveloPment of water stress

The development of water stress in plants is dependent on the

balance between the rate of water loss from the plant to the atrnosphere and

FIG. I

72

Ifetabolic scheme for the mode of

action of CCC by Stoddart (1964).

ËsE@ Possible position of CCC-induced metabolic block

---þ Diversion pathwaY

Porphyrlns r¡--- - -N

Photosynthesis

Soluble rbo hydrates

\\

\\

Storagepolysaccharides

tI

ätural

.//.ú

Ghloroplastproteln I

Strd

polyoac harides

owt h

13

the rate of absorption of l^/ater frorn the soil and its subsequeuL transport

to the evaporating surface. The movernent of water through the soil-planL-

atmosphere continuurn is in response to gr:adients in water potential, and to

soil, plant and atmospheric factors u¡hich influence the development of water

deficits in plants. \^later reaching the root system of a plant depends on

the root absorbing area, soil water potential gradients and soil capillary

conductivity. sì-milarly, the absorption and transport of rvater is dependenL

on plant water potential gradients and root and stem conductivities' Thus

any conclition creating an imbalance in the soil-plant-atmosphere continuum

is likely to cause the development of water stress (Kozlowski, 1968;

Begg and Turner , Ig76; Augusti.n et a-1. 1968). For example a high evaporative

demand usually results in the plant undergoing stress' likervise an excessive

flood. Also the application of PEG Lo the root zone usually results in a

plant water sLress condition.

Although rvater stress occurs when water loss exceeds absorption'

this is not the only condition under whj-ch water stress occurs ' Rather '

t/ater stress is an inevitable consequence of the flow of water along a pathruay

in which frictional resistance and gravitational potential have to be overcome

(Jarvis, Ig75). Thus, all actively growing, transpiring plants experience

some degree of water stress buL Lhe degree to which the stress affects grorvth

and development processes will depend largely on the degree and duration of

the stress and the extent to which the plants can adapt to the sLress (Hsaio '

L973; Begg and Turner , T976). Even in well-watered soils the development

of regular diurnal internal v/ater stress in plants is shown by the mid-day

depression of water potential and the concomittant decrease in transpiration

due to stomatal closure (Kozlowski, 1968)'

As water stress clevelops, many physiological changes take place in

the plant to reduce further loss of v/ater ' or to cope with the increased

vrater loss. Some of these changes will be highlighted below'

T.2.2 Indicators of plant water status

There are a number of methods, direct and indirect, to evaluate

the plant water content. The most wiclely accepted and probably the most

rneaningful crÍterion for measuring plant water status is the water potentíal

designated V (Hsaio, l9l3; *"tß]*rlrrr, +r|t Barrs, 1968)' It depicts the

chemical potential energy of Ufr-ejti"",ru â.td in any situation tlie net hlater

potential is the surn of the hydrostatic pressure or turgor pressure ' the

osmotic or solute potential and the maLric potential which is normally

I4

negligible, as in the follor'ring equaL'i.;onV + VO + Vs + Vn

where V_ is the turgor potential, V the osmotic potential and V_ the matricp " s ' -m ---poter-rtial (Salisbury and Ross, 1969; Milburn, L979; Gardner and Ehlig, 1965).V and V^ norrnall)' are negaLive values and Y_ i,s gene::al1y positive; ho¡ever,s-"pthe existence of negative turgor has been challenged by Tyree (1976). Inaddition to V, measurements of V and VO rnight be useful in assessing plant

S

rr¡ater staLus.

Another nethod for determining plant water status is the RelativeWater Content (RWC) (Weatherly, 1950; Barrs and Weatherly, 1962;

Barrs, 1968). This parameter is the ratio of the actual water content ofthe tissue to the wateï content at fu1l turgor. RWC is related to Y ofthe same tissue and the relationship differs according to species (Connor

and Tunstall, 1968; Sanchez-Diaz and Kraner, I97I; Shepherd, 1976), stages

of growth and also environmental conditions (Knipling, 1967; Augustín et aL.,1968).

Other methods which may be useful include the measurements ofstomatal aperture and resistance and visual syrnptoms such as wilting.

r.2.3 Mechanism of water stress resistance

Plants do adapt to withstand periods of limited soil water ordrought by several means. The ephemerals which produce seeds that can

survive periods of dry r'reather and also cornplete their life cycle before a

period of serious plant hrater deficit develops are referred to as drought

escapers (May and Milthorpe, 1962; Turner, 1979). The drought escapers

normally do not possess any special physiological, biochemical or mor:phological

mechanism to withstand water deficit but survive during periods of water

deficit by rapid phenological development or cleveloprnental plasticity (Jones

et a7.t 1981). Another categor:y of plants endure drought by the loss oftheir leaves. This group includes semi-desert shrubs (Shantz, 1927). These

two mechanisms are the means by which rnany plants which cannot conserve rvater,

but still have to experience periods of dry weather, have adapted to survive(Parker, 1968).

The other mechanisms by which plants can withstand drought involvephysiological, morphological or biochemical responses which enable them toeither avoid or toleraLe the drought. Current thinking distinguishes drouRht

tolerance from drouqht avoidance (Fischer and Sanchez, L979). Drought

avoidance depic-ts the situation in which high plant tissue v/ater potenLial-s

are maintained in the presence of environnrental drought (Hal1 and Schulze, 1980;

15

Begg and Turner, L976) ancl this may be due to morphological and physiologicaladaptations to maintain lvater uptake (increased rooting and increased

hydraulic conductance) and to reduce \¡ater loss (recluction in epldermal con-

ductance, absorbed radiation and evaporative surface). Dr:ouglttolerance on

the other hand involves the ability of plants to pel f orm at lol tissue r'/ater

potentials under r{¡ater deficit. This incorporates the maintenance of turgorby either solute accumulation or an increase in cell rvall elasticity (Jones

et a7. t 19Bl).

As far as this work is concerned, the mechanismsof drought avoidance

and drought tolerance are of much interest and will be discussed further.However, it must be pointed out that whether drought toleranc-e or avoidance

is Lhe nost approprial-e mechanisrn for a given crop is subject to the degree

and duration of rvater deficj-t. and the crop under consideraLion.

I.2.4 Drought avoidance

Drought avoidance, as menti.oned earlier implies drought resistanceat high internal plant water status (Turner, L979; Jones et a7.,1981).

One of the most'important physiological mechanisms by which water

loss is reduced in plants is through stornatal control. Various studies show

that stomata tend to close or do not display maximum opening when plants are

experiencing water deficit. Moreover, there appears to be a threshold water

potential at which the sÈomata close but this varies with the type of plant inquestion (Hsaio, I9l3). Since the stomata are the entry points of C0, intothe 1eaf, the closure of the sLonìata would imply ultinate reduction in assimila-tion and consequently reduced productivity. Due to the importance of thisdiscussion to the present work, the response of sLornata to water stress will be

elaborated later in the text. In addition stomatal resistance, cuti-cu1al-re-

sistance l-o water loss also contri-butes, in some cases very substantially, tothe control of transpirational water 1oss. Blum (1975) reported that, as a

result of increased epidertnal waxes olì an isogenic line of sorghum, there was

a reduction in cuticular transpiration which led to better water status of thatline. In many cases, though cuticular resistance can play an important part

in the control of transpirational water loss, it may be deemed insignificantas compared to stomatal control (Parker, f96BhL Iri addition, nonstomatal

factors in the leaf referred to as ttmesophylltf,ttwallrr resistances nay

contribute to the reduction in transpirational water loss.

Another strategy for reducì.ng water loss is the reduction in the

radiation absorbed. This normally can be achieved by active and passive

¡rf

T1

evitably increases the diffusion resistance to C0, entering the leaf and

ßay, therefore, reduce photosyntl-resis. Therefore, a plant experiencing

water deficj t can conserve water by stomatal closure and naintain high Lissue

water potential at the expense of photosynthesis, or ntaintain a low tissue

wal-er potential but adjust osmoLically which rnay result in partial opening

of the sLomata and at least partially sustain photosynthesis.

The distribution of stomata may differ on the upper and lolver

epidermis of the leaf depending on the species and sometimes environmental

factors. Leaves rvith stomata in both ep idermes are termed ampl-rj-stomatous

whereas those rsith stomata in the lol*¡er epidermis only are terrned

hypostomatous (Meidner and llansfield, 1968). For example, tomato leaves are

considered amphistomatous (Hurd , 1969); however, there are normally more

stomata on Lhe abaxial (lower) surface than on the adaxial (upper) surface.

Under high light intensity the percent increase in the number of adaxial

stomata was higher than the abaxial though the abaxial surface still had a

greater number of stornata per unit area (Gay and Hurd, 1975).

The opening and closing of stonata resulL frorn,,' turgor differences

betrveen guard ce1ls and the surrounding subsidiary or epidermal cel1s (Hsaio,

1973; Zelitch, 1969). Potassium has been found to be the major solute which

accumulates in guard ce11s in light and effects the turgor required for the

opening; the loss 'of potassium from the guard cel1s in the dark results instomatal closure (Raschke , L975; Ehret and Boyer , L979; Hsaio , L973) -

Under water stress conditions, the regulation of stomatal êperbure

is the key determinant of water and C0, exchange. Even in well-watered plants

it has been established that there are diurnal trends in stoinatal conductance

or diffusive resistance depending on the evaporative demand. The diffusiveresistance tends to be high at dawn and decreases Lo a minimum at sunrise; itmay be follorued by a slightly higher resistance aL mid-day in some cases or

the resistance may remain 1ow ti11 sunset and then increase (Jordan and Ritchie,

I97L; l4eyer and Green, 1981). Data available suggest that under conditions

in which water potential is declining, there is a threshold rvater potential

belov¡ which leaf resistance increases sharply indicating stomatal closure.

This threshold differs in dj-fferent plants, it is about -7 to -9 bars in tomato

(Duniway, I97I), -L6 to -18 in cotton (Jordon and Ritchie, I97L; Brown et a7.,

1976) and -11 Lo -I2 bars in beans (Kanemasu and Tanner, L969) " It has also

been reported that stress preconditionì-ng displaced this water potential

threshold for stonatal closure such that stomata closed at a l-or¿er water

potential, and this was morepronounced in the abaxial stomata (Brorvn et a7.,

i'

!

,l

1B

L976i McCree , Lg74). In addition, it has been fottnd t.lìat stomatal

sensitivity to decreasing leaf water potential varies between fj eld conditions

and controlled environments. Davies (Ig71) observed that in both cotton and

soybeans the stomatal sensitivity to declining tvater potential was in the

order of chamber-grown ) greenhouse-gl:or,ün ) field-grown plants'

Adaxialanc]abaxialstomatac]ifferintheirresPonsetowaterstress in some cases. In such cases, the abaxial stomatal diffusive resistance

was lower at the water potential threshold than the adaxial stomaLal diffusive

resistance (Sanchez_Diaz and Krarner, L97I; Kanemasu and Tanner, L969;

Brorvn et a7,, 1916). The behavjour of abaxial and adaxial stomata is also

affected by other external stimuli 1il<e light, darkness and sorne chemicals '

Kanernasu ancì Tanner (rg6g) shor,¿ed that in snap beans, high light intensity

resulted in higher adaxial resistance than abaxial. Nagarajah (1978) provided

evidence that in darkness the adaxial stornata of cotton leaves l^lere more or less

effectively closed while the lower stomaEa were partially open and, in

addition, the reduction in stomatal aperture which occurs with the increase

in age of leaves commenced earlier in the aclaxial stomata and proceeded at

a faster rate than the abaxial stomata' Ùforeshet (1975) reported that phenyl-

mercuric acetate applied to both abaxial and adaxial surfaces of sunflower

plants caused greater closure of the stomata on the adaxial than on the abaxial-

surfaces. Pemadasa (1981) reported thaL the responsiveness of stonata to

light , Coz, KC1 and ABA was substantially greater in abaxial than in the

adaxial stomatal cells of Connelina connunis and that fusicoccin was renarkab1y

effective in stimulatj-ng adaxial stotnata to open more than adaxial ' under

normal conditions the abaxial stomata usually have lower resistance than the

adaxial, which may be explained both in terms of stomatal density and stornatal

opening.

Sincestomatalnovementisturgor-dependentthedifferencesinabaxial and adaxial stomatal response rnay be explained in these Lerms '

Brown et al.. (1976) attribuLed such differences in preconditioned cotton

plants to the fact that the guard cells of the abaxial stomata could lor¿er

their osmotic potentì-al more Lhan those of the adaxj-al stomata ' This is

supported by Pemadasa (1979) who found from histochemical tests that more K*

accumulated in the abaxial guard cells than the adaxial ones ' One can there-

fore infer that, in certain cases, the adaxial and abaxial stomata behave

differentlY and inrlePendentlY '

IthasbeensuggestedthatwaterStressaffectsStomatavjaABAlevels. In the tomato mutant, flacca, which wilts rapidly under lvater defjcit

'I[f','!

t

ttl

19

due to the inability of lhe stomata to close, application of exogenous ABA

to intact platlts reversed this rvilting sympLom (Imber and Ta1, 1970). Inaddition, Tal and lnber (1970) observed that the concentration of endogenous

ABA-lilce substances in flacca r{as about 10 times lower than in the nor¡nal

p1an1-. An increase in ABA levels in plants under \n/ater stress has already

been established (lìsaio, 7973; Vaadia, 1976; Rasmussen, I976; Hiron and

trnlright ' 1973; Mizrahi et a7. ¡ I97O . Apart from tri.gger:ing stomatal closure under

water stress, ABA rnay induce the accumulation of proline (Stewart, 1980;

Rajagopal and Andersen, 1978) r¿hich is important for plant water stressresistance.

Mention is also made of lcinetin as affecting stomatal movement.

Interacting with ABA, reduced levels of kinet.in affected stomatal closure(Bengston et a7.t 7978) and Ta1 and Imber (1970) found that kinetin might. have

a role to play in the excessive openir-rg of the stornata of f lacca tornato,

r.2.6 Chemical induction of stomatal closure

In addition to ABA there are other compounds which, when appliedexogenously to plants, result in stomatal closure and reduced transpiration.Zelitch (1961) found that a-hydrosulfonates induced stomatal closure and

reduced transpirational waLer loss at high light intensities withoutdiminishing photosynthetic C0, assimilation in tobacco. Slatyer and Birhuizen(1964) reported that phenyl mercuric acetate caused a decrease in transpirationin cotton leaves which they associated with the increase in dj-ffusive resist-ance of the leaf to r^/ater vapour transfer due primarily to stomatal closure.Ïn addi-Lion, phenyl mercuric acetate caused a proportionately greater reductionin transpiration than photosynthesis. ZeLiEch and trrlaggoner (L962) reportedEhat phenyl mercuric acetate sprayed on tobacco and maize leaves induced

stomatal closure only on Lhe surface sprayed. They inferred that the sub-stance was not translocated from one side of the leaf to the other; however,

the adaxial stomata closed more readily than the abaxial. Also, the chemical-

reduced transpiration relatively more than C0, assimilation. Mishra and

Pradhan (1968 and L972) showed that phenyl mercuri.c acetate, CCC,.'B-9 and

B-hydroxyquinoline induced stomatal.closure in tomato and thereby reduced

transpiraLion. Moreshet (1975) reported a greater: closure of adaxial sunflower

stornata than abaxial when phenyl mercuric acetate was applied to both sides.

According to Milborrow (1919), phenyl mercuric acetate and farnesol induced

stomatal closure in spinach leaves and this was accompanied by an increased

accunulation of ABA. Ilowever Sabine and Dbr:ffling (fg8f) found that ABA,

phenyl mercuric acetate and farnesol all decreasecl stomatal- aperture buL the

I

t

20

tI

ABA le'el afte:: stomatal closure in the phenyl mercuric acetate treated

Ieaves was not signifì-cantly greater in Spi.nacia and CotnrneTina. They

challenged Milborrowrs (1979) proposition that phenyl rnercuric acetate-induced

closure of stomata was meclj-ated tlirough ABA. Squire and Jones (1971) re-

ported that phenyl nercuric acetate also rerluced cot fixat.ion in the mesophyll

cells, and this inhibition of mesophyll photosynthesis jeopardises the use of

phenyl mercuric acetate as a commercial antitranspirant.

T.2.7 Drought tolerance

The importaDce of maintenance of turgor: pressure for the grorvth

of plants has been emphasized by Flsaio (1973)rand Hsaio et a7' (11916)' tutgBff="tt

influences many of the physiological, biochemicát and morphological processes

in the plant. Therefore, for a plant under water stress to be able to

perform rvel1, turgor pressure must be maintained as water potential declines '

There are Ewo mechanisms by which this can be accomplished; a lowering of

osmotic potential (normally referred to as osnotic adjustmenL or osmoregulation)

or a high tissue elasticitY.

r.2.7.r OsnorepuTation

Under conditions of water stress where the plant water potential

(V) is declining, the plant can maintain its turgor potential (Vn) if the

other cornponents - osmotic potential (Y") and matric potential- (Vr) also

decrease (V = Vì + V" + Ym). Since, as already mentioned, matric

poLential is normally negligible turgor can be maintalned under declining

water potential if the osmotic potential is lowered - The lorvering of

tissue osmotic potential in plants may result from the concentration of

exi-sting solutes during dehydration or by the uptake or int'ernal production

of solutes in the cell. The former <1oes not maintain turgor but the

latter - lowering of osmotic potential as a result of accutnulation of solutes

does (Hsai.o et a7.,.7916; Jones et a7.t 1981; Turner, 1979). The

capacity Lo adjust osmotically is different in different plants ' Turner

(Ig74) found that sorghum adapts better than maize and tobacco; it has

lower osmotic potential at fu11 turgor and this is supported by Sanchez-

Diaz and Kramer (1973) who observed larger changes in turgor preþsure in

sorghum than maize during the development of water stress and after re-

watering. Stress precondi-tioning results in better osmotic adjustment as

reported by Jones and Turner (1978). Though sorghum can adjust osmotic-

ally better than other crops, mention has been 6ade of osmotíc adjusLment

II

Ì

2l

in cott-on (Cutler ancl Rains, L91B), and sunflorver

(Turner et a7., 1978; Jones and Turner, f9B0)'

Thediurnalpatternofosmoticpoterrtjalrvithrespecttothediurnal changes in water potential, has been discussed by Hsiao- et a7'

(Lg16). l,lorking rvith mai,ze, these authors reported that the lotvering

of r.¿ater potential at noon clue to higl-r evaporative demand \{as accoflrpanied

by a lowering of osmotic potent.ial, and the diurnal variation of osmotj-c

potential laggecl behind that of water potenti al. This diurnal variation

in osmotic potential maintainecl turgor despite the rapid decline in water

potential at mid-day and therehy sustained growth'

Several major solutes such as free amino acids, soluble sugars '

carboxylic acids, inorganic cations and anions together are responsible

for the osmotic adjustment in plants. In wheat apices and enclosed

leaves, osmotic potential in the first few days of stress was mainly due

to an increase in the contenL of ethanol-soluble carbohydrate but later

increases in carbohydrate concentrations and amino acids, mainly asparagine

and proline, rnade the major contributions. In cotton, cutler and Rains

(1978) reported that sugars made only a sma1l contribution to the osrnotic

potential but potassium, nitrate and malate contributed substanti-ally'

According to Jones et a7. (1980) the decrease in osmotic potential at full

turgor: in fully expanded sorghum leaves at a moderate level of stress was

accounted for by an increase in the concentration of sugars (glucose and

sucrose), potassium and chloride. In sunflorver, however, half of the

decrease in osrnotic potential at ful1 turgor h/as accounted for by increases

in the concentrations of the inorganic ions, poLassium, magnesium, calcium,

nitrate and free amino acids. In partly expanded sunflower leaves ex-

posed to severe stress treatment, Lhe inorganic anions, chloride and

nitrate and to a lesser extent carboxylic acids (principal]y aconit'ate)

and free amino acids made a si-gnificant contribution to the dqcrease in leaf

osmotic potenEial at fu1l turgor. In four tropical pasture plants' as

reported by Ford and \nh-lson (r98r), the inorganic ions, sodium, potassiurn

and chloride were tile most impor:tant solutes involved in the alteration of

bulk t.issue osmotic potential in the stressecl leaves though proline and

betaine accumulated differently in different species '

Osmotic adjustment has been found to influellce stomatal- closure

under \^,ater stress condit--ions. According to Brown et a1' (f976) the

abaxial stomata of cotton leaves showed a lower water potential t'hreshold

for closure than the adaxial stonata and this was attribr'ited to the guard

li

23

Zebrowshi (1966) rvho found that CCC--treated tomato plants survivcd cold

temperaLures rvhich kj-11ed the unLreated plants. CCC, Phosphon and AM0-1618

induced sal-t tolerance Ín soybean and enabl-ed the treatecl plants to rr'ithstand ,

rsiLhout visible injuries, amounts of commercial fertíIizer, 5-10-5, Lhat

killed the untreated plants (Marth ar-rd Frank, 1961). E1 Damaty et a7. G964)showed an increase in tolerance to saliniLy and drought by CCC treated wheat

plants.

The resistance of planls to drougl-rt and salinitl', as a result oftreatment with growth retardants, especially CCC, has been quite rvell docu-

mented; however, the mode by which the grorvth retardants effect this resistance

is still noL clearly known. Tt rvould not be surprisì-ng if, in differentplants, the mechanism by which CCC confers drought resistance was differentsince CCC per se, affects different species and even different varieties ofthe same species, differently. Plaut et a7. (f964) found a decrease intranspiration rate in bean plants treated with CCC and a decrease in top/rootratio (i.e. an increase in root/shoot ratio) r'¡hich might have contributed tothe survival of the CCC-treated plants under conditions of water stress. Inwheat, CCC promoted root growth as contained in Humphriesr report (1968), and

this involved an increase in the amounL of roots at all soil dept-hs which

would help to sustain Èhe uptake of water from the soil under limited soilwater condition.

CCC has been reported to induce stomat-al closure or inhibitstomat,al opening. Mishra and Pradhan (1967 and 1972) reported that foliarspray of CCC resulted in a reduct-ion of stomaLal width and reduced trans-pirational water 1oss, and delayed wllting of tomato under soil moisture

stress. The reduction of stomatal aperture and an increase in stomata

density due to CCC treatment in cowpea has been found by Imbanba (1912).

Lovett and Campbell (1973) also reported an increased nuniber of stomata per

unit area and increased diffusive resistance in the leaves of sunflower treatedwith CCC. Pi1l et aJ.. (1979) reported that CCC applied foliarly and as soildrench to plants under NHI nutrition increased the leaf diffusive resistance

4and reduced the rvater deficit caused by the ammonium fertilizer.

E1-Fou1y et a7. (1971) found that CCC applied to cotton under water

stress significantly incr:eased protein nitrogen and chlorophyll content ofleaves and also decreased the loss of yield due to \,rater stress but the

changes in cotton yield were independent of those which occurred in chlorophylland protein nitrogen content. The protection of protein under drought

conditj-ons is also supported by CCC-pretreatecl bean plant having more protein

24

nitrogen in the leaves tl1an the untreated planEs, uucler drought (Kharanyan'

1969). Marth ancl Franlc (f96f) observed that t-he salt tolerance induced by

ccc, Al"10-1618 and Phosphon in soybean was not entirely due to reduction in

leaf area. They observecl that mites, which are sucl''ing insects' rnultiplied

more rapidly on leaves of the untreated plants suggesting a developrnent of

chemical and physical changes. singh et a7. (1973) reported that ccc

enhanced the accumulation of free prolj-ne j-n the organs of t¿Lreat under osnotic

shock though ccc did not have any effect on the decrease in rvater potential'

The accumulated proline disappearecl more rapidly from the CCC-treated plants

than the controls when the stress was relieved. They therefore suggested

that the elevaterl concentrations of proline generated in the CCC-treated

plants may play a direct role in supporting renewed gro\"/th once planL water

potential increased.

RoberLson and Greenway (197.3) reported that application of ccc to

young wheat and maize seedlings reduced transpiration as a result of reduced

leaf area, and increased root/shoot ratios. The CCC-treatecl plants did not

show any increase in tolerance to internal water deficits induced by osmotic

shocks or desiccation, and ccc did not affect Etre uptake or rnetabolism of

1abelled glucose. It was therefore inferred that ccc increased the drought

resistance of young maize and wheat plants only by delaying the onset of

severe internal \n/ater deficit, i.e. by drought avoidance' rather than in-

creasing the plantrs tolerance to internal \^Iater deficits' Plaut et a7'

(1964) drew a similar conclusion that ccc and 8-995 might increase drought

avoidance in bean plants. Halev y (T967 ) did an extensive study with bean '

tomato, cotton and gladiolus. He applied different grouLh retardants at a

wide range of concentrations as foliar sPray, soil drench or into nutrienE

culture mecliurn rvith various osmotic valués or under irrigation regimes '

Studying transpiration, stornatal opening, osmoLic potential' water saturation

defj-cit and anatomical and morphological features, he found that none of

these factors showed a consistent correlation rvith the effect- of the chemicals

on drought resistance. He was of the opinion that the effect of the grolfth

retardants on increasing drought resistance might be relaLed to their effect

on delaying senescence; the basic influence of these chemicals being to slol

the breakdown, uncler water stress, of nucleic acids and proteins'

25

CIIAPTER IT

MATERIALS AND I"IETI]ODS

II.1 PLANT MATERIAL

Throughout this work one variety of tomato, namely rGrosse

Lisset was used. This variety was suppliecl by Arthur Yates and Co' Pty'

Limited, and its selection was made solely on lts availability.

TI.2 PLANT GROKITH ENVlRONÌ"IENT

Initial experiments were carriecl out in a glasshouse but the

majority of experiments were in a controlled-environment growth room' and

mention will be made in the text as Lo whether the plants were grown in the

glasshouse or in the controlled-environment growth room'

Theexperimentsintheglasshousemostlytookplaceinthesummerwhen day temperatures were continually high; however, an automatic fan con-

trolled the Eemperature to about 27"C t 2oC during the day and temperature

was about I7"C ! 2"C at night. There was no supplementation to natural light

except that at that time of the year the glass was whitewashed to limit the

excessive solar radiation.

Thecontrolled_environmentgroruthroomhadhighpressuresodiumlamps (8/400 warr), fluorescent tubes (10/60 waLt) ""d lj.jÎdescent

bulbs

(L2/60 watts). The total líght intensity was BO0 pE.m-'S-' and the sodiurn

lamps, fluorescent tubes and incandescent bulbs contributed 857" ' IO7' and 57"

respectively to the total light f1ux. Day temperature was 24"C t 1"C and

night temperature was lBoC I 1"C. Daylength was maintained at 16 hours

Iight, and I hours darkness.

II.3 CULTIVATION OF PLANT

seeds L¡ere pre-germinated in Petri dishes lined with rnoistened

lrthatman No.1 filter paper (15 cm) overlayed with moistenedt\Ki*"ip"='l fin"

grade wipers and the Petri dishes were kept in darkness by wrapplng with

aluminium foil. The filter paper and the Kimwipes I wiper were kept moist

Ï/ith distilled water whenever it was necessary. In 9-10 days later the

germinated seeds with the radicle and plumule separared but the seed coat still

attached, and the tr,¡o cotyledorÌs separated, were transplanted; normally 4-5 to

26

a pot containing tl-ìc rooting medium. The plants tvere laLer thinned to one

per pot for uniformity in size before the CCC application.

Plastic pots rangi-ng from 102 mm to 152 mm (4 ins to 6 ins) indiarneter rvere used. The rooting media used rvere as follows:

(i) Cornpost based recycled soil (ldaite Institute) ruith nutrientlevel up to John Innes I

.

(ii) InternaLional grade concrete sand - coarse mined sand

washed to remove clay (clay 5%).

(iii) 1:1:1 Peat/Perlite/Vermiculite mixture.

In all cases, a known weight of the rooting mediurn underlied by a known weight

of tree bark to facilitate drainage, h/as used.

In the experiments where plants were grov/n in sand or peat/perlite/vermiculite mixture, mineral nutrients were supplied by watering with Hoaglandrs

solution (Hoagland and Arnon, 1938) daily. With the recycled soil, Hoaglandrs

solution was supplied as a supplementation - twice a week.

II.4 PRBPARATION AND APPLICATION OF CCC

Cycocel 1004, obtained frorn Cyanamid Australia Pty. Ltd., with an

active ingredient of 1009/1 was used. In all Lhe experiments, CCC leas pre-

pared in Hoaglandrs solution and the quantity required for each experiment r,ras

prepared the same day that j-t was applied.

As mentioned earlier, the method of application of CCC affects ttre

responsiveness of plants (Bhattacharjee et a7.¡ 197I; Humphries, 1968). In

tomato, CCC applied as a foliar spray has been effectj-ve (Pisarczyk and

Splittstoesser, 1979) as well as when applied to the rooting medium (I^/ittwer

and Tolbert, 1960; Abdalla and Verkerk, I97O; Abdul et al..t I97B). However,

hlittwer and Tolbert (1960) observed that foliar spray, though effective,wasnot long lasting as compared to soil treatment. Moreover, in a limited space,

drops of the CCC solution spreading onto plants not meauL to'be treated is not

impossible. Therefore, in this work, it was thought appropriate to apply tLre

CCC only to the rooting meclium. The volume applied in each case. is specified

in the text.

METFIODS OF STRESS IMPOSITIONII.5

stress was imposed by with-holcling water and allowing the available

2l

soil moisLure ro be clepleted. Alternatively polyethylene glycol (PBG M'\'ü'

4000) was used to induce water sLress'

i^/here water stress was induced by with-holding water to the

rooLing medium, the pots were ernbedded in polythene bags tied at the end to

the lower portion of the stem to prevent direct evaporation from LTre rooting

medium. Naturally, plant water deficit results when the rvater absorbed and

transpired by plants is not replenished. This, in fact, sets the soil-pJ'ant-

atmosphere continuum in an inbalance (Kozlolski, 1968) sj-nce obviously ' the

rate of \'rater loss will override the rate of water absorption as the soil

water is dePleted.

PBG is a well knorvn osmoticum. Husain and Aspinall (1970) and

Singh et a7. (1973) have used PEG (M.W.4OOO) to induce stress without any

specific toxic effects. Lesham (1966) and Lawtror (1970) have reported toxic

effects due to PEG.

In this work PEG (1\/1.\^1" 4000), used to induce stress' was applied

in Hoagland I s solution to effect the required plant water potential ' In the

two experiments where pEG was used, abnormal symptoms developed especially in

the leaves and this will be discussed beloiv'

II .6 l'{EA 0F PLANT \4rATER STATUS

II.6.1 Leaf water Potential

Leaf waLer potential was measured with the spanner-type thermo-

couple psychrometer and also with the pressure bomb (Boyer, 1969; Barrs' 1968)'

With the pressure bomb technique, the petiolus of the leaflet was sealed in

the chamber such that iL protruded about 5 rnrn above the seal' The blacle of

the leaflet was subjected to pressure inside the chamber and at the point that

the xylem sap appeared at the cut surface the pressure build-up was stopped

and Lhe water potential read off the pressure gauge. In order to rninimize

error in this operation the increase in pressure was so controlled that the

pointer of the pressure gauge travelled aL a reasonably moclerate speed ' In

addition, the chamber was lined with moist filter Paper to reduce the dehydra-

tion of the leaflet due to the pressure'

In the psychrometry, half of the I eaflet blade rvas placed against

the inner wall of Lhe charnber and sealed with the thermocouple plug ancl then

placed in a water bath aL 25"C t 0.001"c. An equili-bration time of 2-3 hours

28

was a1lowed. The deflections from a movíng charL recorder were used to

conpute the r^¿ater potential after comparison with standard calibration values

using known molar solutiorrs of sodium chloride' Since the psychrometer used

hacl 24 chambers the water potential of the samplecì leaf let \ras measured ttvice

wiLh each half of the blade in two different chambers and the average value

taken.

II.6. 2 Leaf osmotic Potential

osmotic potential was determinecl with spanner-type thermocouple

psychrorneter(Barrs,1968).Throughouttheexperiment.s,thesametissueused for the determination of water potential r¡as frozen betlveen sheets of

filter paper and thawed for about 30 minutes and then reloaderl into the chamber

and the same procedure for the psychrometric measurement of water potential

(section II.6.1) followed. Though many workers freeze their tissue in the

chamber, this method can ruin the chamber due to repeatecl freezing and

tharving. on the other hand, freezing the tissue in the open could lead to

precipitationofatmosphericwateronthetissue.Inordertoavoidthese,the tissues \¡/ere ftozen and thawed in between sheets of filter papers and

bef ore reloacling, all the chambers vrere vriped clean of all dr:ops of water '

II.6.3 Relative rvaLer contenL

The rerative water conl-ent (RWC) was deLermined using leaf discs

1.5 cm in diameter. The fresh weight of the leaf discs were Laken imrnediately

after pur-rchlng them out and then the leaf discs were floated on distil'led

water under diffused light r.or 4 hours (Barrs and weatherly, 1962) ' The

discs were then blotted dry with filter paper ancl the turgid weight takert

prior Lo oven .drying at BOoC for 24 hours and rneasuring clry weight' The R\trC

was then calculated frorn the formula (Barrs ' 1968):

Fresh weis,ht I)rv rveiqht x 100RWC = Fully turgid weight Dry weight

TI.7 I'IEASUREI'{I1NT 0F GROLITH PARA}4ETERS

II.7. 1 Plant }leight

The height of plant \,/as determinecl by measuring the heig,ht of the

stem from the soil surface to the apex'

means of a piece of string trailed along

In inLact Plants, thi-s was done bY

the stem from the soil leve1 to the

29

apex and t-he corresponding lengt.h of the string measured on a rul-e. Inexcised p1 ant the stem height \,r/as measured d j recLly on a rule af ter excisionof the leaves.'

Tr,7.2 Leaf Area

Leaf area \,/as measured on a Paton electronic planirneter. The area

of the leaflets rvas actually measured without the petiole since the device

used could not handle the petiol.e. The leaf lets \,/ere put one af ter the olheron a transpar:ent conveyor and passecl between a line of photocells and a lightsource at a constant speed and total area read on a digital readout. The

planimeter \{as equi-librated for at least 30 rninuLes and calibrated before use.

II.l .3 Fresh weight

The freshvreighing these organs

petiole were excised,

leve1 and immediately

away the soil or sand

weight of the leaves, sLem and root rvere determined by

on a lvfettler (P1210) balance. Leaves including the

and rveighed immediately. The stem h¡as cut at the soilweighed. Roots were extracted by carefully vrashing

and blotting dry prior to weighing.

II.7.4 Dry weight

Leaves, stem and roots l/ere separately put into paper bags and

oven-dried at BO"C for 48 hours and then weighed.

II. B LEAF D]FFUSIVE RESISTANCE STOMATAL COUNTS AND TRANSPIRATION

II.B.1 Leaf diffusive resistance

The leaf diffusive resistance r{as measured wi-th a Li-Cor DiffusiveResistance Meter, model LI*60 (Lambda Instruments Corporation) with a horizontalLambda nodel sensor (Kanemasu et a7,t 1969). The whole assembly is normally

referred to as a leaf diffusive resistance porometer. The diffusive resistancewas measured by inser:ti-ng the leaflet in the sensor such that the sensor cup

was on the surface to be measurecl. The time required for a given amount ofwater vapour to diffuse into the sensor cup and be absorbed by the humidi tysensing element was recorded. The resistance v¿as computed by comparison withcalibrat-ion values

"

II.B.1.1 Caiibration of diffusive resi-stance Poroneter

Calibrations of the pororneLer were clone in a constant temperature

30

room at 20oC and in the contro1led-environment grorvt-h room ryith a

tenperature of 24"C + 1oC, rvhere the majoriLy of the experiments were

carried out. The calibration proc.eclure developed by Kanenasu et a-Z "

(1969) was followed.

The resistance for the cali-bration l\¡ere derived frorn a set ofholes (of the same size as the aperture of the sensor of the diffusiveresistance pororneter) on the upper plate of a pair of acrylic plates.Strips of Whatman No.1 filter paper (24.0 cm), 1 rnm thick, were moistened

and placed on the acrylic base plate such that the ends di.pped into a

distilled \^/ater reservoir on both sides. These strips thus served as

waLer wicks. A coarse chromatographic paper cut to the size of the platewas moistened and placed on the filter paper rvj-cks. A hlhatman No.1

(24.O cm) filter paper also cut to the size of the plate, r./as moistened

and placed over the moist coarse paper and run over with a glass rod toremove excess water. The upper acrylic plate rvhich is the resistance platewas then fastened over the upper filter paper. The sensor cup of the poro-

meter, always kept dry by storage in a dessicant, was attached to the

resistance plate. The rvhole assembly was then allowed to equilibrate forabout I hour and during thÍs time dry air was intermittently pumped

through the cup. With the sensor cup over a blank space on the resistancep1ate, the meter of the porometer was adjusted. The instrumenl- was then

calibrated for transit times between Lwo meter readings, 30-70 using theI'Humidity-2'r leve1 of the meter with a s1-op watch. Starting with thelowest resistance (i.e. the open space), several readings were Î-aken foreach set of resistance holes-open (Lo/s), 60, 30, 15 and B (Table II.1) inorder of increasing resistance. The relaLionship between the time lapseAt ancl resistance (sec..r-1) are presented in Fig.II.l. for the constant20oC temperature room ancl the 24oC + 1"C controlled environment growth rc¡on.

II.8.1.2 Measurenent of Teaf diffusive resistance

Before taking any set of readings, the meter of the porometer was

switched on, adjusted to full scale (100) and allorved to equilibrate for30 minul-es during which tirrre dry air was pumped through the sensor cup from

time to t.ine. Prior Lo a measurement dry air v/as pumped through the sensor

cup such that the nieter read 20 or 1ess. The sample leaflet (usually the

Lerminal leaflet) \^/as iDserted into the sensor carefully making sure thatthe leaflet was not crumpled in Lhe sensor. The time lapse for the pointer

of the meter to travel from 30-70 was recorded using a stop rvatch. The

temperature of the leaflet h/as also recorcled from the meter and later con--

TABLE II.1

31

Calibration values of the diffusive resistance porometerat 20oC and 24oC

A). Calibration at 20"C

Number of resistance holes

t (secs)0pen(L/o) 60 30 15 B

57 .r

7LL,2110. 0LO7.6110.8109.3108.7110.5110.7108.9109.6

181 .8178. Ir80.5179.2rBl .0r77.3178.9180. 7

178.5I79.I

64.3s6.959.259.356. B

58.257.959. I58.7

7

5B

04B

29B

2

434T4343424243424I43

35,226.727.r28.529.328.727.228.227.527.9

Mean t (secs) 28.63 42.83 58.75 r09.73 r79.5L

ùlean resist?nce 0.61 3 .27 6.53 13. 07 24 .5O(sec cm -)

B). Calibration aL 24"C

Number of resistance holes

t (secs)0pen(L/s) 60 30 15 B

128.5r49.3r39.4r52.5131 .0146.8150. II5I.2r49.7148.5

76,888.286.386.480.981 .090,786.583.287.1

502

033759B

454s4443454547434B4s

34.933.332.O30.432.635.033.534.634 .035.2

40435021

65

2323232222222I242323

lulean t (secs) 22,80 33.55 45.42 84.7I r44.76

Mean resistçnce O.B0 3.13 6.27 12,54 23.5I(sec cm -)

32

FIG. II.1: Relationship between the time lapse

( t) and diffusive resistance in the

calibration of the porometer at 20"C

and 24"C

(r) 24"C (^) 20oC

The straight lines were fitted by linear regression

analysis.

20"c 24"C

Y=6.44x + 22.O5

R=0.998

Y=5.42x + 16.33

R=0.998

i¡

I

I

I

200

r80

ló0

r40

120

C)

Sroo

ã

ó0

40

20

80

Â

5 10 15 20 25

Diffusive Resistance (sec emÐl

JJ

verted to oC. Tnjtial experience witli t--he porometer: indicat-ed that- after

the f irst tneasurernerrt, and especially on the adaxial surf ace, a second

measurenjent on LIte sane surface of the same leaflet gave a higl-rer diffusive

resistance. Therefore, on a given leaflet the resistance of the aclaxial

surface was firsL measured bel:ore the abaxial surface. Care was also

taken nol- to breathe on the leaflet at Lhe time of measurement.

II.8.2. 1 StonataT counts

The initial atternpts to peel off the epidermal layer were very

frustrating since the layer v¡as delical-e and would not peel off easily'

Therefore the method of Sampson (1961) was used for rnaking imprints of the

leaf surface. Leaf discs were punched from the blacle of the leaflet and

floatecl on distj-lled water to prevent rvilting. Sjlicone rubber was spreacl

on the surface after blotting the disc dry with filter paper and this

hardened within 15 minutes. The hardened plastic tvas gently lifted away

with forceps, washecl in detergent and then rinsed in distilled rvater.

This was thoroughly dried with filter paper and then placed flat with the

impressed surface upwards in a dessicator containing si'lica gel for 15

minutes. Clear varnish (transparent naj-l poU-sh) v/as sPread over the dry

and undisturbed impressed surface of the silicone rubber and inmediately

replaced into the dessicator for about 15 minutes. The varnish replica

was separated from t-he silicone rubber and the number of stomates/unit

area counted under a light microscope.

II.B.3 Transpiration

Total transpiration in intact planLs was determj-ned gravlmetricall y.

Pots were embedded in polythene bags, the ends of which were tied around the

lower portion of the stem. The whole system - pot (polythene bag) with plant

- vras weighecl initially and later after a given tirne period. The transpira-

tion rate was calculated from v¡ater loss from the intact plant for a known

period of time per unit l-eaf area.

Transpiration rate in the excised leaf was determined by excising

the leaf and cutting the petiole under water and then sealing it in a plastic

bottie (as shown in Appendix T), The water loss in a known time interval

was expressed on a unit leaf area basis.

-l ¿-t

rr.9 CTIEMICAL PROCI-ÌDURES

II.9.1 DeLermination of Proline

The determination of free proline Ín the leaves was done follorving

the nethocl of Singh et a7. (1973). Prior to the extractiou and measurenent

of free proline, the leaf tissue rrras lrozen in liqr-rid nitrogen and freeze-

dried. A knorvn weight, normally about 200 rng, of the freeze-dtied leaf

tissue together \^/ith 1.59 of Decalso-I' resin was placed in a Dual glass

homogenizer and homogenised in 5 ml of methanol : chloroforrn : water (!l\'lC

L2:523) at. roorn tempel:ature. The homogenate was decanted into a centrifuge

tube and 5 ml distilled water and 3 ml chloroform added to break the enulsion.

The mixture was then shakerr thoroughly on a centri-fugal shaker and centrifuged

at 2OOO rpm for 5 minutes. The upper aqueous layer was then transfer::ed to a

boiling tube containing 5 rnl glacial acetic acid and 5 ml of freshly prepared

ninhydrin reagent (125 mg ninhydrin : 3 ml glacial acetic acid : 2 ml 6l"f

orthophosphoric acid), and then placed in a boiling water bath for 45 minutes

with a glass marble covering the boiling tube to prevent excessive evaporation.

After cooling to room temperature, 5 ml toluene was added and then shalcen

vigorously. The two layers rvere allowed to separate for 30 rninutes and the

optical densiLy of the toluene layer read at 520 nm. The concentration of

proline was estimaLed from a standard curve (0 to 100 ug prolì-ne).

II .9.2 Extractj,on and assay of quaternary amrnoniumcompounds using the N.l'1.R. technique

Fresh tomato leaf tj ssue (2-5 mg) was homogenized in 10 ml

nethanol: chloroform: water (MCh/ 12:5:3) in a glass centrifuge tube rvhich

sat in an ice bath during the homogenization. The grinding head of the hono-

genizer was washed with 5 nl of distilled hlater into the original homogenate

and centrifuged at 3OOO rpm. The supernatant (l"le0H/water phase) was removed

and the pH adjusted to a range of 6-7 and then loaded on to a Dowex column,

followed by distilled water until 100 ml of eluent had been collected and

this was discarded. The Dowex column consisted of 59 of Dowex 5O\{/H+/50-1OO

nesh/2% cross-linkage. Before each use, it was washed with about 25 m1 of

BN I-ICI and then f lushed with distillecl water unti 1 the pH of the eluent was 5.

After the first elution with distillecl water, the second elution of the Dowex

column was 4N HCl and 1OO ml was collected. This acid eluent was dried under

vacuum using a rotary evaporator and a water bath at 50"C - 60"C. The dry

residue was washed with 5 ml of distilled water and redried. The lasttraces of HrO were removecl by drying under a steady stream of N, for about

35

5 minutes. The sanple was then dissolved in 0.6 ml DrO. Care was talcen

for tl-ris dissolution since the dry sample had spread all over the evaporator

flask but still had to be dissolved in the 0.6 ml DrO. 0.4 ml was removed

and transferred to a 5 mm Nl,{R tube. The result from the NMR tvas thus nulti-plied by 1.5 as a volume correction factor. A reference standard, 20 u1 of

tert.-butanol was added to the NMR tube. The spectra was then run and the

integrated areas obtained. The areas of the compounds of interest \,'¡ere ex-

pressed as percentages of the t-BuOH peak area and the amount of each compound

was determined from a standard curve.

rr. L0 EXPERIMENTAL DESTGN AND STATISTICAL ANALYSIS

fi randomized compleLe block design was used in all the experirnents

and the number of r:eplicates used in each experiment is specifiecl in the text.The pots, in which the plants h¡ere growing, were rotated intermittently to

minirnize positional effect.

DaLa were analysed by the analysis of variance method and depending

on the factors involved either the randomized complete block analysis or the

factorial analysis was used.

36

CIÌAPTER III

RESULTS AND DISCUSSION

III. 1 CCC AND I^JATBR STRESS EFFECTS ON WATER

STATUS AND GRO\-/TH OF TOI'{ATO

III.1.1 Introduction

It has often been reported that CCC enhanced the ability of some

plant species to withstand drought. The very early rvork by llalevy and

Kessler (f963) showed that CCC increased the drought tolerance of bean plants

and since then a substanti=l amount of work has been done relafing CCC and

drought resistance.

Tomato, as compared to other plants (brjgalow and mulga) is very

sensitive to drought (Connor ancl Tunstall, 1968) and it was, therefore, worth

testing CCC on it under drought conditions. As mentioned earlier, CCC

affects different species and even different cultivars of the species

differently (Cathey, L964). It was therefore necessary to carry out some

initial studies on the growth and water status of the tomato cultivar used

and hor.¿ it was affected by CCC under normal conditions and under rvater stress

conditions. Thi-s, as reported here, took the form of the:

tif'tI

(i)(ii)(iii)

Establishment of an effective concentration of CCC

Effects of this concentration on the grorvth of tomato

Age or stage of grorvth of the plant at which this con-

centration could be applied.

].II.I.2 Effects of different concentratlons of ccc and

water stress on water potential and growth

TT.I.L.2.I Introduction

Plaut et a1. (1964) reported that different concentrations of CCC

produced varied results on bean plants under various irrigation regimes '

In potatoes, Dyson (1965) found that higher concentrations of CCC recluced

Stem gl:owth more than lowel: concenLration, and similar results were

obtained by AbduI et a7. (f978) in tomato. However, high concentrations

could be toxic to the plant. This has been found in Norway Spruce

(Picea abies) on which CCC at 300 mg/1 was toxic (Dunberg and EIJ-ason'

Ig72). It therefore becane necessary for a satisfactory 1eve1 of CCC

I

I

i

37

to be deternined for this worlc since, under a given set of experimental

conditions, too high a concentrat-i-on could be toxic whil e too low a

concent.ration could be ineffective.

Initj al observaLions indicated that concentrations below 500 ppm

rvere relatively ineffective buL concent--r:ations between 500 ppm and

1000 ppm were most effective. Those above 1500 ppm pr:oduced toxiceffects mainly in the form of yellowing of the leaves starting from the

mid-vein and spreading towards the margin of the leaf blade. Plants

which received 1500 ppm could recover and grow normally but at 5000 ppm

the plants did not recover. Thus, in this experiment., concelìtrations of

0, 750 and 1000 ppm were tested.

The tomato seeds \4rere pregerminated as described in Section II.3and transferred to I52 mm (6 ins) pots containing 2 kg of sand each rvittr

100 gm of tree barlc pieces to facilitate drainage. The plants were

initially watered hrith half strength Hoagland I s solution and later withfull strength as they grerv bigger. CCC (750 and 1000 ppm) was applied

in Hoaglandrs soluLion at 200 ml/pot to 3-4 weeks old plants and the

untreated plants received an equal volume of Hoaglandts solution. üiater

stress was imposed by with-holding water 2 days after the CCC treatmenL

and the pots were embedded in plastic bags to reduce evaporation.

hlater potential of the uppermost fu11y developed leaf (3rd or 4th

leaf ) was rneasured with the pressure bornb. Four days after rvith-holding

water the plants were harvesLed and the growth parameters measured. The

experirnent was carried out in the controlled-environment growth room withconditions specified in Section II.2, and the treatments \{ere replicated4 times.

TII.I .2.2 Resul ts

There \¡¿ere no significant differences between the water potentialof the plants under field capacity but, with time water stress resulted ina decline of water potential in all the treatments. Hotvever, both the

750 ppm and 1000 ppm CCC treatments maintained significantly higher ttaLer

potential (i.e.less negative) than the untreated plants under water stress

(Fig. III.f.1). The 1000 ppm CCC rreatnent maj-ntained the highest water

poLential in the course of the v/ater stress developnrent. By the fourth

day after: vrith-holding water, the water potenti-al- of the unLreated plants

*

.J

¿l

))

((

))

((

tr'.

- (r)(tr)

FIG III.1.1:

Control (0 ppt)

750 ppm

1000 ppm

(F.C. = Field Capacity)

3B

Effects of different leve1s of

CCC pretreatment on water Po-

tential during 4 daYs of with-

hoì ding watcr.

F. C.Stress

F. C.Stress

F. C.Stress

oo

A

^

i(

IïI

þ

12

fo

II L.S,D 5%

I

.n¡-o.oI

=.g.l-,CoPoo.l-o*t(u

=

6

4

2

o

o1234Time After W¡th-holdi¡rg

Water (daYs)

I

Iri

39

under water stress had reached -10"75 bars rvhile the 750 ppm aucl 1000 ppm

CCC-treated plants had attained water potentials of -8.5 bars and -6.65

bars respectively. At this point the unLreated plants showed synPtoms

of wilting - the leaves had collapsed and the leaflets \ì/ere rolling up

buLthe 750 ppm CCC-treated plants had their leaves beginning to collapse

with no rolling up of leaflets while the 1OOO ppm CCC-treated plants did

not show any of these symptoms as shown in Fig. III.I.2. The behaviour

of tomato leaves under rvilting conditions tlust be comrnented on at thisjuncture. Under: water deficit, the first visible sympton is the collapse

of the leaf petiole and this normally occurs at a water potential c¡f

between -B bars and -9 bars. This is then followed by the rolting up of

the leaflets as the waLer stress increases in severity and all these

syrnptoms were delayed by the CCC treatmenLs especially at 1000 ppm.

Both levels of CCC significantly reduced the height of the plants

6 days after the treatment and \.{ater stress also reduced the height of tÌre

untreaLed plants but not the CCC-treated planÈs (Table III.1.1). The

total leaf area and the number of leaves per plant were unaffected by CCC

alone but were decreased by water stress though the CCC-treated plants

maintained higher leaf area than the untreated plants under water stress.

The dry weight of the whole plant was only significantly reduced by \^/ater

stress and this reduction could largely be accounted for by the reduction

in the leaf dry vrej-ght since the stem and root dry weights were unaffected

by water stress.

III. f .2.3 Discussiot't

The maintenance of higher !/ater potential under water stress due

Eo the CCC treatments agrees with the work of De et a7. (1982) rvho found

that in two varieties of wheat both seed treatment with CCC and CCC

applied to 45 days o1d plants resulted in the treated plants maintaining

higher water potential than the untreated plants under drought conditions

in the field

Theheightoftheplantk¡asretardedbytheCCCtrea.tmentandsimilar results were obtained rvith tomato by Abdalla and Verkerk. (1.970).

There was no further retardation in the height due to waEer strðss in the

CCC treatments but water stress recluced the height of the untreated

plants. Leaf area and nunber of leaves were not affected by CCC alone

40

FIG. ITI.1.2:

From rightto left:

Photographs showing CCC-treated and

control plants under well-wateredcondition and under stress induced

by h¡ith-holding water. The CCC-treatedplants hrere supplied with 1000 ppm CCC.

ccc F.c.Control F.C.CCC stressedControl stressed

The Control stressed plant at the extreme left showssymptoms of wilting as described in Section IIf .I;2.3,whereas the CCC-treated and stress plant next to itdoes not show the w-ilting symptoms.

1

tvJ ¡tv

tI t Þ4v

) Ir

-L'

:I

,.!

l.¡.'r

..-l!$

r

.tf.l

r \,, ..

I*.

{

'¿Ê

--+ b

Ð

I I

TABLE ITI.1.1: Eff,ects of different levels of CCC and water sLress on growth characteristics after 4 daysof with-holding r^rater.

Height ofplant (cm)

Leaf area(¿'2)

Number ofleaves

Dry wei-ght (g)Stem Root Whole plantLeaves

F.

F.C. Stress F.C. Stress F.C. Stress F.C. Stress F.C. Stress F.C. Stress F.C. Stress

0

750

1000

L.S.Ð. 5z

CCC

Stress

CCCìíStress

19.76

17.L5

17.47

17.52

16.78

17.24

1 .36

i .38

1 .40

0.48

0. 84

0.93

6.42

6. s0

6.75

5.67

5.33

5.84

0.60

o.73

0.70

0.46

0.49

0.48

0. 18

0.24

0.20

0.20

o.24

0.2r

0.09

0. r3

0.11

0.11

o.r20. 11

0. 87

1. 10

I .00

0.76

0.84

0.77

0.117.27

1 .04

0.03

0.36 0.07

F.C. = Field capacity.

0.28

42

but were by water stress. The drastic reduction in the leaf area of

the untreated plants by tvater stress could be attributed to the rollingup of the leaflets. The reduction in the nunber of leaves pel: plant

in all the treatments, due to rvater stress, could have resulted from the

senescence and subsequent dying of the lot,rer older leaves, especlally

the first 1eaf. The dry weight of the leaves but not the stem or root

rvas reduced in all the treatments by water stress despite the fact that

the CCC treatments maintained higher leaf water potential. Similar

results were found by Gates (1955) rvith tornato under \,/ater stress where

the stress affected the dry weight of the leaves more than the other parts

and both moderate and severe stress reducecl the dry',',eight of the leaves

to the same extent. This possibly suggests that, in tomato, leaf growth

is more sensitive to l^/ater stress than the other parts and once water

begins to become limited leaf growth is drastically affected.

III.1.3 The effects of 1OOO ppm CCC on leaf, stem and root growth

III. 1.3.1 Introduction

From the previous resulLs (Section III.1.2.2) the 1000 ppm CCC

treatment maintained high water potentì-al under water stress and ' on that

basis , l\¡as chosen f or all subsequent experinents.

Generally, CCC retards the stem growth of plants and in some

cases the leaf and root growth as well. This CCC-inducecl inhibition of

growth has been observed in tomato (Pisarczykand Splittstoesser, 1979;

Abdalla and Verkerk, 1970). This, therefore, necessitated a study of the

growth of the tomato cultivar used for this work when treated r*¡ith 1000

ppm CCC.

pregerminated seeds were grown in 1 kg recycled soil (\rJaite

Institute) in tZt rnm (5 ins) pots and half strength Hoaglancl's solution

was supplied to the plants twice a week. 1000 ppm CCC was applied as

150 mt/pot soil drench when the plants were 3 weeks old. tr{ater stress

was imposed in some of the plants by with-holding water .10 days after' '

the CCC treatment. The height of the plants was measured in both the

rvell-watered plants as well as the sLressed plants with tirne' To another

set of plants the 10OO ppm CCC was applied rqhen they were 4 weeks old and

43

leaf growth and tl-re fresh and clr:¡' weights of the various orgaiìs \lrere

studied in well:rvaLered plar-rts 5 days and 16 days after treatment r'¡ith

CCC. The experj.nents were cal:r-i-ed out in the controlled-environtnent

growt-h rooni with the sane concli.tions as stated in SecLion II .2" The

\rarious treatments \,/ere repli.cated 3 tilnes.

III. I .3.2 Resu-lts

The height of the plant was not significantly reduced by CCC in

the well-waterecl plants until the 6th day after the CCC treatnìent

(Fig. III.1.3) and rher:after the CCC-induced retardation in plant height

became more ancl more significant. By the 16th day, the height of the

CCC-treated plants under well-watered conditions hacl reached only 697. of

their untreated counterparts. hlater stress significantly reduced the

height of the untreated plants but there was no further significant re-

duction in the CCC-treated plants due to v/ater stress.

In the second batch of plants, 5 days after the CCC treatnent

none of the parameters measured had been affected by CCC (Table III.1.2).

Sixteen days after the CCC treatment, the height of the p1ant, total leaf

area, the fresh and dry weights of leaves and stem had been significantlyreduced by CCC but not the fresh and dry weights of the root. Thus, the

reduction in the shoot fresh and dry weights accounted for the reduction

in the whole fresh and dry weights in the CCC-trealed plants. The rootf

shoot ratio on fresh weight basis but not on dry weight basis rvas

significantly higher j.n the CCC-treated plants.

A study of the leaf area and leaf length of the individual leaves

with respect to their position on the stem showed that 5 days after the

CCC treatnent, there were no differences in eiLher the leaf area

(Fie. III.1.4a) or rtre leaf length (Fig. ITI.1.4b) at any position

between the CCC-treated planLs and the untreated controls. By the 16th

day after the CCC appl.ication, the leaf area of the 3rd leaf and all the

leaves higher Lhan the 3rd hacl been reducecl by CCC rvl-rereas the length of

the 5th leaf and all the leaves higher than the 5th had been reduced by CCC.

Control (0 pp*) -

CCC (1000 ppm)

44

Retardation effects of 1000 ppm

CCC and rvater stress.

@ ) F.C.O ) SEress

F.C.Stress

FIG. III.1"3:

((

- (^ )(a )

24

20

11IL.S.D. 5%

4

2

1

1

Eo*¿Ê(r-o.rFo+asu'o-I

I

4

o

I

tSfress

o246 8 10 12 14 16

Time after CCC application (daYs)

TABLE III. 1 .2:

45

The effects of 1000 ppm CCC on varj-ous grot+thcharacteristics of r,¡ell-rvatered tomato p1ants.

5 davs after CCC treatrnent 16 davs af ter CCC treatmentCharacteristic Control CCC L. S. D.

(57")Control CCC L.S.D

(57")

Height of Plant(.*) ô

Leaf Area (dr')2t.876.37

26.9610.179.06

46.20

3.96o.470.284.7r

o.250.06

19 .606.32

26.4r9.928.31

44.64

o.240.06

3.04o.57

4.r2o.971 .60

O. BBo.240.041. 13

40.9012.T3

59.3328.6516. 81

ro4.20

8.602.390.91

11.90

0. r90.08

22.r7B.9s

49.96L7 .64r7,2084.10

3,74r.75

5.383.422.O2

10.63

1 .000.430. 11r.44

Fresh weights

LeavesSternRoothlhole plant

(Ð

5.50

Drv weishts

LeavesStemRootWhole plant

Root/Shoot ratio:Fresh weight basisDry weighL basis

(Ð3.750.430.264.45

71

09

4553BBB1

03o2

0.010.006

00

2609

00

FIG. TII.1.4:

5 days afterCCC treatment -

16 days aft,erCCC treatment -

46

Bffect of 1000 ppm CCC on (a) leafarea and (b) leaf length, withrespect to leaf position, 5 days

and 16 days aften the CCC

treatmenL.

( O ) Control(^) ccc

( o ) Control(^) CcC

a28

2.4

.î LOEo(ú

I t.óC'

((toJ 1.2

0.8

o4

a

L.S.D. 5%

r0

3ó

32

28

24

?20oG,

g tó-g

6-3 t2

o

b

2468Position of leaf on stem

2468Posítion of leaf on stem

I L.S.D. 5%

Ii

II

I

I

I

1

I

I

I

4

o r0

47

III.1.3.3 Discussi.on

The height of plants was not significanLly reduced by CCC in the

well-watered plants until after about 6 days (Irie. III.1.3). This

agrees with findi-ngs of Abdalla and Verkerk (1970) in whose rvork CCC re-

tarded the lieight of tomato plants af ter 7 days. In addi-tion \{ittwer

and Tolbert (1960) observed that the shortening of internode length in

CCC-treated tomato plants occurred withj-n 5-7 days after treatment and

van Bragt (1969) reported that CCC applied to the rooLs of tonato in-hj-bited the increase in height of plants 5 days after the application of

the CCC. Generally, work done $/ith CCC shorvs that the shortening of

the height of the plants can be observed about a week after the CCC

application. For example, in r'¡heat, Singh et a7. (1973) observed a

retarclation in plant height by CCC 10 days after the treatment.

From Table III .I.2, leaf area and fresh and dry weights of the

plant organs were unaffected when the height of the plant had not been

substantially retarded by the CCC treatnent (i.e. 5 days after CCC treat-ment). However, once the height had been retarded' as was the case 16

days after CCC treatment, the total leaf area, the fresh and dry weights

of the leaves, stem and the plant as a whole v/ere reduced by the CCC

treatment. In tomato, Pisarczyk and Splittstoesser G979) reported that

2 weeks after the application of CCC, the leaf area and the dry weight of

the treated plants were less than the untreaLed. Contrary to the shoot,

CCC treatment did not have any inhibitory effect on the rooL grotvth and

this resulted in a higher root / shoot ratio, especially on fresh weight

basis, in the CCC-treated plants. Several workers have observed an

increase in the root / shoot ratio due to CCC treatnent. As reported

by Hurnphries (1968), CCC stimulated root growth in wheat. In the present

work the increased root / shoot raLio found with the tomato plants was

not due to increased root growth but rather to the inhibition of shoot

growth without any inhibition of root growth.

By the 16th day after CCC application the leaf area and length

of the 4th and 5th l-eaves respectively, and all the higher leaves had

been significantly reduced (Fig. III.1.4). This suggests that CCC ex-

hibited an inhibitory effect on the gr:owth of the leaves which were stilldeveloping at the time of the CCC application and the leaves forrned after

4B

the CCC application, ancl this may account for ttie overall reduction in

the total leaf area of the CCC-treated p1'ants.

III.1.4 The effects of 1000 pprn CCC applied aL clifferent stages ofgrowLh and tvater st,ress olì vsater potential and growth

I1I.1.4.1 Introduction

Generally, rvork clone to st,udy CCC-induced resistance to drought

has been carried out such that clrought conditions are imposed after CCC

has ef f ectively retarcled growth. This poses the question as to r'¿hether

t.he CCC-induced drougtrt resistance is not nerely due to the reduced

evaporative surface resulting fron gror'rth retardation, which in turn,

controls l^/ater loss and thereby maiutains better water status. This ex-

periment therefore was designed to study CCC and water stress interactj-ons

when CCC had retarded growth and before it had affected growth. From the

previous experinents (Section ITI .I.3',2), CCC retarclecl the growth of the

shoot by about 6 days after its application" This suggested that

differences in the length of time of CCC application before stress

imposition might affect the treated plants differently. Thus 1000 ppm

CCC was applied at an early and at later stages of the vegetative growth

so as to achieve a retarclation of growth and non-retardation of growth

respecl-ively, by the time stress was imposed. This was to ensure that

both the CCC-retarded and CCC-non-retarded plants were of the sarne

physiological age by the time of stress imposition notwithstanding the

complications of the plants being treated with CCC at different stages

of growth and for different lengths of time before stress.

Pregerminated tomato seeds \,rere grown in 1.5 kg recycled soil j-n

152 mm (6 ins) pots in the controlled environment grorvth roon with con-

ditions as stated in Section II.2. llalf strength Hoaglandrs solutioD was

suppli-ed twice a week as a supplementation. 1000 pprn CCC was applied as

200 ml/poL to one batch of plants when they were 13 days old; they rvere

designated |'CCC Earlyt' treatment. The rtCCC Latett treatment comprised

plants treated with CCC rvhen they rvere 25 days old. hlater stress was

imposed by r^rith-holding water from the plants 3 days after Lhe I'CCC Laterr

treatment, (i.". 15 clays after the rrCCC Earlyt' treatment). Just before

imposing the sLress a harvest was rnacle frotn well-watered CCC-treated and

49

untreated plants and a second harvest was nade at the end of the stress

episode (i.e. 9 days after wiLh-holding water). The treatments were

replicated 3 times.

TIT "I .4.2 Resu-l ts

Before the imposition of water stress (i"".3 days and 15 days

after CCC treatment in the "CCC Late[ and "CCC Early" respectively), theI'CCC Early'r treatmenL had retardcd grorvth of both shoot and root, but

increased rooL/shoot ratio, whereas the "CCC Latet' treatment had noL

affected growth (Table III.1.3a).

Under normal well-watered conditiotts CCC did not affect the

water potential (Fig. III.1.5). Flolever, in the rvater-stressed plants,

Lhe non-CCC-treated plants sholed a rapid decline in their water

potentials after the 3rd day of \^¡ith-holding water. This rapid decline

was cleferred by both CCC treatments especially the "CCC Barlyil treatment.

Moreover in the course of the stress, both the "CCC Earlyt'ar-rd I'CCC Late"

treatments rnaintained a significantly higher potential than the non-CCC-

treatment, but waLer potential was still higher in the "CCC Early"

Lreatment than in the trCCC Latetr treatment. At the end of the stress

episode the water potential of the "CCC Early", "CCC Lateil and the non-CCC-

treatments were -15 bars, -15.8 bars and -18.8 bars respectively. The

trCCC Laterr treatrnent retarded growLh of the shoot but not ttre root thereby

increasing Lhe root to shoot ratio 12 days afLer the CCC treatnent (i.e.

9th day of stress) as shown by the I'CCC Late" F.C. treatnent inTable III.1.3b. Also, water stress inhibited the growth of plants in

all the treatmenLs and increased the root/shoot ratio.

IIT.1.4.3 Discussion

Tire lack of clifferences betl*¡een the tvater potentials of ttre CCC

Lreatments and the non-CCC treatment under rvell-watered conditions, but

the maintenance of higher t{ater potential in Lhe CCC treatrnenEs l-han t}re

non-CCC treatment of stressed plants agrees with the results in Section

ITI.l .2.2 (Fie. III.1.1).

The relardation of root growth in the t'CCC Earlytr treatment

before the imposition of water stress (i.e.15 clays after CCC treatment),

F]G III. 1 .5:

ttCCC Early"

-

CCC was appliedplants

ttCCC Laterr

-_ CCC was appliedplants

50

Effects of 1000 ppm CCC appliedat different sLages of grorvth

and water stress on water potential.

to 13 days old

to 25 days old

Control

I'CCC Early'r -

ttccc Latett

(^ ) F.c.( Â ) Srress

))

((

@

o

@o

SCS

F.CStr

F. C.Stress

))

((

22

20 I L.S.Ð. 5%

18

16

14

o2468 10

Time After With-holdi¡rg Waten(days)

II

12

o

I

6

1

4

2

o

Øbo,ClI

-(u.Ftro+toÈbo+.(tt

=

:tI

51

Effects of 1000 pprn CCC applied at different times(Ear1y and Late) on growth characteristics before and

after stress.

TABLE II].1.3:

(a) - Before str:ess impositionHeight ofplant (cr¡)

Leaf area Drv weight (g)(A*2) Lea"es Stem Root

Root/Shootratio

Control-CCC EarlyCCC Late

2t.5614.5420.14

7 .854.64B. BO

3.r41 .953.23

0. s90.4r0.60

0. 150.170. 15

0.770.4so,7 4

L.S.D.s% 2.58 2.64 1.01 0.17

(b After 9 davs of stress)

Height ofPlant (cm) Leaves

Dr 1¡/e htStem ot Root/Shoot

ratio

F.C. Stress F.C. Stress F.C. Stress F.C. Stress F.C. Stress

ControlCCC EarlyCCC Late

L.S.D. s%

cccStressCCCY'Stress

34.6723,6028.00

26.2718.932r.27

2.06L.721 .89

2.L3I.I41 .95

0.190.23

o.20.250. 28

423

444B46

I0I 230

42904t

I.24 0.900.83 0.66r.24 0.94

2.251 .84

0.850.68

o.420.34

o.2ro,26

o.o20.02

al

52

is not consislent rviLh the resul-t in section III'1'3'2 (Table III 'I'2)

rvhere CCC dicl not retard root gr-owth 16 days ¿rl-ter its application'

Thisdiscrepancycouldpossiblybeacc:ounteciforbytlrecliffererrcesit'ttheagesofLlreplantsatwhichCCCrvasapplied.InSeclior.iIII.I.3,2,ccc rvas appliecl to older plants (4 weeks o1cl) rvl-ier:eas it't the present case

the plants in Llre ',CCC Early'' treatment \vere treatecl with CCC at a re-

lativelyyoungerstage(13clayso1d).Tlreincreaseinroot/shootratioin the ,'CCC Earlyil treatment l¡ef ore stl:ess impositi on could possibly be

due to shoot grorvth being retarded more than root grorvth ' The I'CCC Latert

treatment did not have any effect on growth before stress j'nposition (-i'e'

3daysafterCCCapplicati-on)butretardedtheshootgroruthrvitlroutaffecting root gro\{th 12 days after the treatment and' thereby' increas-

ingroot/shootratio.Simi]arresultslverefcundinSectionIII.I,3,2(TableIII.1.2)whereCCCdidnotaffectgrowtl'r5daysafterjtsappl.icationbutr.etardedshootgrorvthrvlthoutaffectingrootgrotvth,16 daYs after its aPPlication '

DespitethemaintenanceofhigherrvaLerpotentialunder\^raterstress growth could not be sustainecl ancl sirnilar results v¡ere found in

SectionIII.1.2.2(TableIII.1.1).ItisalsoevidentthattheCCC_induced maintenance of higher r'¿ater potential under stress is independent

of its growth retardation effect '

TNDUCED IJATER STRESS AND SOIL h'ATER

I

i

). : I'

ITI.2 A COMPARISON OII PEG_

DBPLETlON I]Y hIITiI- IIOLDTNG W ATER COiYBINED WITH CCC TREA TI"IENT

]-IT.2.7 Introduction

Inallthepreviousexperiments,tvaterStresswasinducedbywith-holding waLer from the plants and there has been a repeatable effect of

ccc treatmenl on the treated plantst ability to naintain better water status

under stress. Robertson and Greenway (1973) reported that the grorvth of

ccc-treated maize and wheat seedlings was affected to the same ext-ent as the

untreated plants tvhen subjected to nannitol-inducecl water stress' Data

from the work of singh et a7. (i973) showed that the waLer potential of ccc-

treated wheat plants declinecl to the same extent as that of the untreated

plants under PEG-induced rvater stress '

tI

3

53

It was therefore founcl appropr-iate Lo compare Lhe effects of

CCC on tontato plants uncler water stress conditj'ons induced by soil water

depletion (i.". by rvith-holding rqat-,er) and an osntoticutn. In thj-s section,

the effect of CCC on the water status of tonato truder eiLher PEG-induced

stress or soil water clepletion, and the growth of CCC-treated ancl unLreated

tomato plants under PEG stress were studiecl.

The tomato p]-ants \,/ere grown from pregerminated seeds in 152 mm

(6 ins) pots containing 2 kg sancl. The plants rvere rr¡atered rvith half strength

Hoaglands solution irritially anrl later with full strength ' CCC at 1000 ppnr

was applied (200 rnl/pot) to the treated plants rvhen Lhey rvere 3å tveeks o1d'

trrlater stress was inposed 4 days afLer the CCC treatment. PEG (M.W'4000)

at two levels, -B bars and -12 bars osmotic potential , was appliecl (300 nrl/

pot) in Hoaglanclrs solution daily to the sand medium, to one batch of plants'

In another batch of plants water stress was inposed by rvith-holding water

from the plants; the pots were embedded in polythene bags to minj'mize

evaporation of water frorn the surface of the sand ' The non-stressed plants

were watered daily v/ith fu1l strength lIoagland's solution' Before the

irnposition of stress (i.e. 4 days after CCC treatment), some plants were

harvested and some growth parameLers - heigtrt of p1ant, leaf area ' dry weight

of leaves, stem and roots were nìeasured. Leaf water potential of the upper-

most fu1ly developed leaf was nonitored with the pressure bomb during the

stress period. At the end of the stress episode (i.e. B days of stress),

a second harvest was trade to evaluate the effect of PEG at both levels on the

grol,/th parameters menLioned above. The experì-rnent , which was carried out in

the controlled-environment growth room ' r'¡ith conditions specified in Section

IT.2, was rePlicated three times.

I.IT,2.2 Results

Before the imposition of stress, CCc had not had any effect on

the grorvlh of the Lreated Plants, as shown in Table III.2.I; the height of

plant, leaf area arrd clry weight of leaves, stem and roots had not been

affected by the CCC treatment.

As can be seen from Fig. III.2.I, -B bars PEG and -12 bars PBG

decreased the water pote¡tial, and there was no difference in effect on

either the CCC-treatecl or untreated plants. Soil water depletion also in-

-.,tr!T

!

i

I

iI

if

xil

'.iI

f,'ì

I

III'l,l

,l

I

I

t

TABLB III.2 .1 :

5/+

EffecLs of CCC on various gro\'/th characteristicsbefore the imposition of stress.

Height ofplant (cm)

Leaf qrea Drv weight (g)(¿r2) Lea"es Stem Root

Control 22.60

2r.20

5.82

5.73CCC

3 ,37 0. 81 0.68

3.24 0.84 0.72

rttI

L.S.D. s% 2.30 t.26 0.90 0.41 0.38

,t

I

II

I.

f

It

I{i

FIG. ]TI.2.1:

Cont::ol -

CCC

55

Effect of CCC on water poLentialunder PEG-induced sLress and

soil moisture depletion induced

by with-holding water.

(e) F.c.( O ) Soil water stress( â ) -8 bars PEG stress( g ) -12 bars PEG stress

F.C.Soil water stress-8 bars PEG stress-12 bars PEG stress

AAocl

((((

))))

I

I

18

16

L.S.D. 5% I

678

PEG -12 barsi=-¿

Soil

Soil*CCC

PEG -8bars

F,C.

14

12

10

I

6

4

2

o

U'Lo.clI

(sEgc)+toÀt-o+)GÈ

o1 234sTime of Stress(days)

56

ducecl a decrease in rvater poLential, buL, contral-y to the PliG stress, the

CCC-treaLed plants naintained higher water potenti-als than the unLreated

plants.

By the thjrd day of stress, the PEG-treated planLs showed

symptorns of toxicity, these symptoms \{ere nore severe in the -12 bars PEG

treaLment. The margins of the leaflets were chl-orotic and rvi lty and the

symptonìs spreacl s1or,u1-y towarcls the mid-vein. The central portions of the

leaflet larnina, holever, remained non-chlorotic but were abnorrnally darl<

and shiny green and appeared water-loggecl. The petiole and sten, on the

other hand, appeared turgi d and even by the end of the stress they appeared

more turgid than those of thc plants subjected to soil water depletion.

This PEG-induced toxicity synclrome affected the CCC-treated ancl non-CCC-

treated plants to t-he same exLent.

Data frorn Table III.2.2 indicates that at Lhe end of the stress

episocJe PEG, at both levels, had significantly decreased grorvth; height of

plant, dry weíght of leaves, sten and roots had been clecreased in the CCC-

treated and untreated pJants alike. Uncler field capacity, however, CCC

slightly decreased sl-root dry weight but increased the root dry weight

thereby increasing root/shoot ratios (Table III.2.2).

III.2.3 Discussion

CCC did noL have any inhibitory effect on the growth of plants

4 days after its application (Table III.2.l). As discussed in Section

III.1.3.3, CCC does not retard the growLh of tornato until about a week

after its application.

When tonato plants are subjecLed to soil water deplet1on, CCC

enhances their ability to retain higher v/ater potential, and this has been

the case in al1 previgus experiments. On the other hand, when subjected

to PEG stress, water potential decreasecl to the sarne extent in the CCC-

treaterl ¿rnd non-CCC-treated plants. Singh et a7. (1973) reported a similar

effect of PEG in that Lhe v¿ater potential of CCC-treated and uut-reated wheat

plants were clecreased to t-he same extent. ft is not clear why CCC-treated

plants behave differently wl-ren subjectecì to the trvo different methocls of

stress, but secondary effects of PEG may be involved '

TABLE TTI.2.2t

57

Effect.s of CCC and PEG-induced stress on various grohlthcharacteristics .

Height ofplant (cm)

Dry weight Cg)Leaves Suem Root

Root/Shoot ratio(dry wt. basis)

Leaf(dm

area71

Control F.C.

ccc F.c.Control stress 1

(-8 bars PEG)

CCC stress 1

(-8 bars PBG)

Control stress 2(-12 bars PEG)

ConLrol stress 2(-12 bars PEG)

L.S.D. 57"

ccc

SLress

CCC*Stress

33.20

25.60

23.33

22.77

23.O7

22.57

I .93

2.36

TT.22

8.76

1 .53

8.00

7.06

3.50

r.97r.62

I .31

2.t63. 19

1.36

1.56

1. 10

I.20

o.22

o.37

0.28

0.31

0.30

0.31

0. 14

3.41 I.52

2.41 L.2O

2.s6 r.29

0.90

1.78

0.31

0.s4 0.43

5B

Toxic effects due to PEG have been found in certain situations.

Leslram (1966) reportecl that PEG rvas Loxic to Pinus haTepensis seedlings.

According to Larvlor (1970), the presence of PiÌG j-n leaves causes necrosis

rvhich rnay ulLi-mately lcill the leaves. Hoclgson et a-7-. (1949) reported

that pEG treatment of tomatoes causecl marginal- wilting of l-eaflets wl-rj-le

the leaflet certtres, petioles and stelns still had high water content

cornparable to the controls. They also found that a large portion of the

PEG taken up by the plants was in the leaflet malgins whj ch wilted ' Joyce

(1980) observed that i¡ radish, PEG-i¡cJuced leaf margi'n necrosis led to un-

realistically high water potent1al measuremenLs in the pressure bornb;

this he attributecl to the ingress of air into the leaf through xylem exposed

to the aLmosphere due to the necrosis. Tllus, the PEG-induced toxicity

synrlrome reported in this tsork is not unusual and the symptoms observed were

sini-lar to those reported by Hodgso:a et a7. (i949). Nonethel.ess, the fact

that CCC treatment could ¡rot naintain better rr¡aLer status under PEG st::ess

and that toxic symptons showed in the CCC-treated plants, as well as the

untreated ones, s¡ggests that the devastating effects of PBG on the tonato

plants could not be ameliorated by the CCC treatnent '

pEG decreased the growth of both CCC-treated and untreaLed plants '

Results from section III.1.4.2 indicate that ccc could not sustain growt-h

under soil water depletion" This, therefore' suggests that irrespective

of the source of water stress, growl-h under water Stress is not sustained

by CCC.

For all subsecluent experimenLs ' water stress rvas induced by with-

holding water. This method was adopted due to PEG toxicity and the lack of

effect of ccc on water potenLial in the PEG-stressed plants.

III.3 CCC AND \^TATER REI,ATIONS 0F TOMATO

III .3. I Introduction

previous results revealed that CCC-treated plants, when under

water stress imposed by with-holding water, maintainecl higher water poterrLial

than the untreated plants . This, t.heref ore , called f-or a f urther sttlcly

into Lhe water relat.ions. In this section, the effects of CCC on \^/ater

potential , relative ,,^rater content (R\^lc), osnotic potenti.al, r-urgor potenLial

and their inter-relationships, particularly when growth had not been retarded

by ccc bef ore sLress imposition, rvi1,l be d j-scussecl .

59

Tf.I.3.2 Relative \'/ater content'

rrr .3.2 . 1 14ethod

Pregerminated toniatcl seeds \,/ere g]:own íl I27 mm (5 ins) pots

containing I kg recycled soil and half strenglh Hoagland's solution

supplied trvice a rveek. 10OO ppm CCC (150 ml/pot) was applied to the

soil rvhen the plants v/eïe 3å rveelcs old. Water stress rvas inposed by

with-holding rvater from the plants 3 days after CCC application' \'/ater

potential and R\tiC of the 5th leaf (the uppermost fully devel'oped leaf)

were measured as outlinecl in seclions If.6.1 and II.6.3 respectively'

The experiment was carried out in the controlled-environment growth room

with conditions specified in sect.ion II.2, and was replicated three times'

III.3 .2.2 Resu-l ts

lrlater potential was higher (i.e. less negative) in the CCC-

treated plants than the untreated plants under water stress but there

v/ere no differences in the well-rvatered plants (Fig' III'3'1)'

similarly, RI¡JC was higher in the ccc-treated plants than the

untreated plants under water stress and this difference was more pro-

nounced after the 3rcl day of stress (Fie. III'3'2)' By the Bth day of

the stress, RWC had dropped to 577" and 53% in the CCC-treated and un-

treated plants respectively. There L/ere no significant differences in

the RtrtC under rsell-watered conditions betrveen the CCC treatment and non-

CCC treatment.

The relationship between R\n/C and water potenti,al (Fig. III'3'3)

showed a pattern which fitted a linear regression analysis. . However,

the regression coefficients of the CCC and the non-CCC treatment were not

significantly different (0.939 anrl 0.935 respectively), neither were

these different from the regression coefficient of Lhe poolerl data (0'934)'

This indicated that data from both CCC treatment and non-CCC treated

fitted the same linear regression. Thus, at a given water potential

R\4lC did not differ in the CCC and non-CCC treatment or in other words '

a uni-t fall in R\^jC decreaserl water potentj-al to the same extent in

treated and non-treated Plants.

FTG. II].3.1:

Control -

CCC

60

Bffect of CCC and rvater stress

on water potential.

( ¡. ) F.C.( À ) Stress

SCS

F.CStr

@

o((

))

I

20

18

16

14

12

10

I

6

4

2

o

ØL(tÍ¡I

G.Ftro+too.l-oP(U

=

o2468Time After W¡th-holding

Water (days)

!-.t.o. u*

FIG. III.3.2:

6L

Effects of CCC and rvater stresson RhlC.

Control -

CCC

@

o

AA

((

((

))

))

F. C.Stress

F.C.Stress

I] :tr L.S.D. 5%

òq

ocoooCÚ

=o.l(ú

o(r

r00

80

ó0o

40

20

0

o 8

Ti¡'ne After With-holding Water (days)

642

62.

FIG. I]].3.3: Relationship betrveen RhlC and water

potential (moisture release curve).

( @ ) Control(a) cCC

The straight line was fitted by a linear regressiouanalysis.

= 0.2-5X - 28.L1

= O,934

Y

R

Retative Water Content (9o)

o 20 40 60 80 100

a

-2

-4

-8

-10

72

Øl-(ú.o

¡

ñ{:Eo+.oÀbo*tG

È

-6

^

O

L

44

-16

63

III.3.3 Osmotic potential and turgor potential

rrT .3.3. 1 Method

Tomato plants were grolin from pregerminated seeds planted in

I27 mn (5 ins) pors conraining 1 kg 1:1:1 peat/petlite/vermiculitemixture. The plant-s v/ere continuously supplied r+ith half strength

Hoagland I s solution initially and then l-ater with full strength as they

grerv bigger. 1OO0 ppm CCC rvas appliecl as 150 ml/pot when the plants were

about 4 weelcs old. Tmposition of stress l'/as by l^/ith-holding water 3 days

af ter the CCC treatment. Sone of the plants \,/ere re\¡Iatered af ter 6 days

of water stress. Leaf water potential and osmotic potential were

measured by the psychrornetric method as the stress progressed and also

during the recovery period. Turgor potential was deduced fronr the

difference between osnotic potential and t'¡ater potential. The experiment

was carried out in the controlled-environmenL growth room with conditions

as outlined in Section II.2 and was replicated three Limes.

III.3.3.2 Resu-lts

Similar to the previous experiments CCC treatment resulted in the

maintenance of higher water potential under water stress, but after re-

watering both the CCC-treated and untreated plants recovered at the same

rate (Fig. III.3.4). I^lithin 12 hours the water potential of a1l l-he

treatments had reached normal values.

During the period of stress, osrnotic potential of the non-CCC

treaLment was lower (i.e. more negative) than the CCC treatment

(Fig. III.3.5) and t.here v/as no difference between the two treaLments

under well-watered conditions. Upon stress alleviation, the osmotic

potent.ial of the CCC-treated and untreated plants increased at the sarne

rate but did not reach normal values as quickly as the \,Iater potential.

The relal-ionship between water potential. and osmotic potenti-al, as

illustrated in Fig. III.3.6, shows that at a given r,¡ater potential, the

osmotic potential was the same for both treatments; hovrgve::, stress

al-leviation altered the relationship at lower osmotic poLentials with

no effect of CCC.

64

i?rc. l-rr ,3 " 4: Effect of CCC on \,/ater Potentlzrlunder stress and recoverY.

Control - ) F.C.) Stress

CCC F.C.Stress

DoLLed line -

water potential under recovel:y.

((

@

o

A,

A))

((

18

16

14

12

10

L.S.D. 5%

\

Rewatered

t

I

I

6

4

2

o

tÍtL(E¡tI

GFço+.oo.Lo+.(g

B

Ill

il

ll

tl

tl

lr

IIIIlu

0 2 4 61Time of Stress and

Recovery (days)

65

FTG. IIf.3.5: Effect of CCC on osmotic potentialunder stress and recovery.

Control - F.C.Stress

CCC F.C.Stress

Dotted line -

osmotic poLential under recovery.

@

o

A

^

((

((

))

))

18

16

14

12

10

I

Rewatered

L.S.D. 5%

ll

illl

tIII

.t,¡-G.ctI

-oEEo+too.

.9*.oE1r,

o

I

6

4

2

o

o24Time of Stress andRecovery (days)

1

-ttll

i

66

RelaLionship between water potentialand osmotic potential.

Þ-.c.S Lresslleco ve ry

( r, ) F.C.( A. ) Stress( Q) Recovery

Lines fitted after Morgan (I977a).

FIG. III.3.6:

Control -

CCC

ooD

(((

)))

I

c! t

Osm

otic

Pot

entia

l (-b

ars)

T@ I

oo

o c{ I

\

\o ri-T T

\o I \ 'a

\... 'lò of

tr

o

?T G -o I€ I

o .,= = O@

+tl o o. LO O

T+

t o =e{

T

a\{

1tr or

a\\

R.

s T \o r\

U

,æ+

g-È

.-i>

¿l

67

Turgor potenLial, deducecl from the difference between osmotic

potent,ial and water potential, did not differ significantly T¡etween the

Lwo treatments uncler \\rater stress or under well-watered conditions

(Fig. III.3.7). Turgor lvas recovered very rapidly in the stressed plants

after rewatering, though the rate of recovery was the samê for the tlvo

Lreatnents. At a gì-ven water potential turgor pressure was the same in

rhe two Lreatments (Fig. III.3.B).

rrr.3.3 .3 Discussion

The maintenance of higher water potential due to CCC treatment

under water stress (Figs. III.3.1 and III.3.4) supports earlier findings;

however, upon rewatering, CCC did not have any effect on rate of recovery

of the water potential which could possibly be due to the rapidity wi-th

which the plants recovered.

The lower osmotic potential in the non-CCC-treated plants under

stress (Fig. III.3.5) may be attributed to the fact that during the stress

episode, the non-CCC-treated plants suffered a more severe internal water

deficit than the CCC-treated plants as manifested in the water potential

data (Fig. III.3.4). This less lowering of osmotic potential in the ccc-

treated plants is reflected in the ccc treatment not siEnificantly affect-

ing turgor potential under stress (Fig. III.3.7). Upon stress alleviation

osrnotic potential increased at the same rate j-n both treatrnents but because

osmotic potential did not reach norrnal values as rapidly as h/ater potential

(Figs. III.3.4 and III.3.5) there was a drarnatic increase in turgor

potential ruhich dj-d not differ between the two treatments during ì:ecovery

period (Fig. III.3.7). Sanchez-Díaz artd Kramer (f973) also found that,

in maize and sorghum, turgor potential returned to values higher than

norrnal after rewatering.

CCC did not affect the relationship between hlater potential and

osmotic potential suggesting that both the CCC-treated and non-CCC-

treated plants reached zero turgor at the same water potential (Fig'

III.3.6); however, stress alleviation altered the relationship without

any effect of CCC such that zero turgor might be reached at a lower water

potential. In plants which can adjust osmotically to resist water stress'

osmotic potential is so lowered that zero turgor is reached at a lower

Ìilit'

I

t

6B

F]G. I]I.3 Effect of CCC ou turgor potenLial

under stress and recovery.

Control F-. C.Stress

CCC

Dotted line _ turgor potential under recovery "

@

o((

))

l-. c.SLress

(a)(¡)

t

tI

lr

I o o. -ñ U'

a4. - o g, at

, o o. :E o o o o -{ CL

A) (t,

oTur

gor

Pot

entia

l (b

ars)

l\)À

o.

o l\) ÀÐ o q A

¡ r+ o \ o a.

<- tr cn I i¡ I

F]G rrI 3. B:

Control -

CCC

69

Relationship' betrveen \'/ater poten-

tial and turgor Potential.

F.C.StressRecovery

(a) F.c.( a ) Stress( Q) Recovery

(((

@

otr

)))

oç, o

A

-4-20

u

6

4

2

{c(oo-ï,ooJ

EoA).aq,

ootaA

oo

Ao AoÀo Âo

-r ó -t4 -12 -r 0 -B -6

Water Potential (-bars)

70

water potential (Morgan , 1977a; L977b) ' Thus, sorghutn can adjust

osmotical ly better than maLze and tobacco because its osmotic potential

is lorvered more (Turner, Lg74). CCC, therefore, did not enhance Lhe

planLs ability to adjust osrnotically but str:ess alleviation did' This

may explaj-n the lack of difference in turgor potential at a given \\rater

potential (Fig. III.3.B) due to ccc treatment and the high turgor

potential values during recovery. The lack of CCC-induced osmotic

adjustment is also supported by the fact that CCC did not alter the re-

lationship between \{atet: potential and Rl'/C, i.e. the moisture release

curve (Fig. III.3.3). According to Jones and Turner (1978) osnotj-c

adjustment and the decrease in elasticity in sorghurr under stress caused

a shift in the moi-sture release curve such that there l¿as a small

decrease in R\dc per unit decrease of water potential.

IIT.4 CCC AND ACCUI'1UI,ATI ON OF PIìOLINE AND

QUATERNARY A DS

III.4.1 Introduction

The previous results indicated that the prolonged survival of the

CCC-treated plants did not involve CCC-induced osmotic adjustment. Singh et

a7. (1973), however, obsetved that CCC treatnent protnoted proline accunula-

tion under water stress. Since proline accumulation has been suggested as

a possíble indicator of stress or drought resistance (Singh' et a7" 1972;

Palfi and Juhasz,LSTI), the effectaJ ccc on proli-ne accumulation in tomato

uncler water stress was explored and, in addition, an attempt was made to

ascertain whether quaternary ammonium conpouncls ivere accumulated that may be

related to plant stress resistance.

Proline was extracted by the rnethocl outlined in Section 1I.9.1

in two separaLe experiments. In both experiments, the plants \{ere grown in

tr-27 nn (5 ins) pots cont'.aining 1 kg recyclecl soil ancl were supph-ed rlrith half

strength Hoaglandts solution twice a week. VJater stress was induced by

with-holding water from the plants 3 days after CCC application' fn l¡oth

experiments, horqever, proline was extractecl from t[e uppermost ful1y

developed leaf of CCC-treateri and untreated plants under rvatø. stress and

71

well-rvatered conclitions. Wat--er potentia] of tl-ie sanple leaves was recorded.

In yet another expe::iment--, Nf{R tecl-rniques, outl.ined in Section II.9.2, vrere

used to study the quaternary anmoniurn conpouncls rvh'icll accumulated in the

leaves of both the CCC-treated anrl untreated plants; leaf rvater poLctrlial

was again measurecl and the plants !/ere grohrn under similar conditj-orìs as

described above.

TII.4.2 Results

The data fron the first experiment in r,/hj-ch proline was extracted

are presented in I'ig. III.4.1 and Fig. III.4.2 lor water potentl-al and proline

accumulation respectively. Ccc-treated plants maintained hi gher waLet

potentj,al than the r:ntreated plants urrder r{ater stress induced by with-holding

water (Fig. III.4.1). The untreated plants under \{ater stress accumulated

more proline than the CCC-treated plants until the 6th day after with-holding

water when the CCC treatntent overtook the non-CCC treatment, Lhough CCC did

not significantly increase the amount of proline accumulaLed by the Bth day

of stress. The proline content remained the sarne in the non-stressed plants

over the stress period and did not differ between the CCC-treated and non-CCC-

treated plants (Fig, :l.TI-.4.2), The results fron the second experiment

showed that CCC treatment maintained better \{ater status under stress than the

non-CCC treatment (Fig. III.4.3) and this agrees rvith the data from the first

experiment (Fig. III.4.1). The trend of proline accumulation in the secoud

experirnent (Fig. III.4.4) was si-milar to that of the first experiment

(Fig. III.4.2) except that the amounts of proline were less in the second

experiment.

The NMR specrra (Figs. III.4.5 and IIT.4.6) indicated that CCC

and choline were the major quaternary arnrnonium compounds detected as was

proline in the leaves of the CCC-treated plants (Fig. III.4.5) while choline

and proline alone were detected in the untreated plants (F-ig' III .4.6).

From Table III.L¡.I, the proline content in the leaves of the unLreated plants

uncler stress was higher than in the leaves of the CCC treatnent but waLer

potential was lower in the untreated plants (-11.20 bars) than the CCC-treat'ed

plants (-9.80 bars). The amount of choline was affected neither by CCC nor

stress. The CCC content rvas hi gher in the well-rvatered Lhan the stressed

plants.

FrG. rtr.4.1:

Control -

CCC

l2

Effect of CCC and rgater: stresson water potential.

) F.C.) Stress

F.C.Stress

((

@

o

A

^))

((

20

18

16

14

l l.t.o. u*

12

o

I

6

4

2

1

.t,¡-o.oI

G'.{;tro+too-l-o*toB

o

o

o2468Time After W¡th-holding

Water (days)

FIG TTI.4,2:

Control -

CCC

73

Effects of CCC and water stresson proline accunulation.

F. C.Stress

( ^â ) F.C.( À ) Srress

@

o((

))

ï I f It.t.D' s%

-c.9)o

=oo)Eo.EofLo)5

8

6

4

2

12

t0

ô-ô0

0 4 6 I

Time After With-holding Water(days)

^

2

FIG III .4.3:

Control

CCC

7lt

Effects of CCC ancl water stresson water potential.

(e) F.c.( O ) Stress

SS

F.C.Stre

(¿)(^)

I

í'¡

I

i

20

18

16

14

12

II I L.S.D. 5%

^

10

I

6

4

2

ú,l-o,clI

G''pÊo+¿oo.l-c)þtñts

o

o2468 10

i

i

Tlrne After with-holding water (days)

FIG. III.4 .4:

Control -

CCC

75

Effects of CCC and rvater stresson pr:oline accumulatj-on.

F.C.Stress

((

eþ

o))

SESF.CStr

(a)(^)

J

ì

!.

I

6

I I fl.s.D. s%A

Ò

I 10

-c.9o3

oo)EoEofLo)Þ

4

2

ôo

o 2 64

Time After With-holding Water(days)

FIG. III.4.5:

76

NNIR spectrum of an extract of a

CCC-treated plant I s leaf, shorving

choline, CCC and proline peaks.

Water

Choline r7

Standard

ccc

Prolin

t.

4ppm

I

FIG. I]I.4.6:

77

NMR spectrum of an extract ofa non-CCC-treated plantrs 1eaf,shorving choline and proline peaks.

Water

Choline

Standard

Proline

{

ppm

TABLE III.4.1:

7B

Proline, choline and CCC contentof leaf estimated by the NIIR

technique.

Waterpotential(-bars)

d we1 ne Choline

t

Control F.C.

Control sLress

ccc F.c.

CCC stress

4.00

11 .20

4.20

9.80

4.Ls

6.78

5.24

0.63

0.69

0.78

o.62

3.40

L.O7

L.S.D. 57" 2.O5 1.00 0.22 r.23

79

III.4.3 Discussion

The rnainLenance of higher rvater potentÌ-al , under \vater st--ress

condÍ-tions, due to CCC treatment (ltig. IIT.4.1 and Fig. III.4.3), supports

the repeatable effect of CCC-induced enhancenent of water stat-us uncler

water stress induced by with-holding r¿ater frorn the plants, as found in the

previous experinents.

Overall, CCC dicl not increase the plantsr ability to accurnulate

proline in the leaves under water str:ess. The initial 1ag of proline

accumulation in the CCC-treated plants may be attributed to the delay in

the onset of severe inLernal water deficit, as manifested by t-he water

porential data (Figs. III.4.1 and III.4.3). This suggests that the accumu-

lation of proline is dependent on the r^,,ater potential, as has been suggested

by Aspinall and Paleg (1981). Though Singh et al.. (1973) found that CCC

promoted proline accumulation in wheaL, tlie water potential declined to the

same extent in both the CCC-treated and non-CCC-treated plarrts as a resultof the PEG-induced stress. However, in addition to using different tisstte,

Singh et a7. (1973) imposed stress by a different method.

Choline levels were high in the tomato leaves but neither CCC

nor sLress promoLed its accumulation (Table IfI.3.1). This supports the

fÍnding of Mayr and Paxton (1962) who observed that the main quaternary

ammonium base extractable fron untreated tomato plants v/as an unidentiflablesubstance which was readily converted to choline. The occurrence of CCC

in the Leaves of the CCC-treated plants (Tabte III.3.1) suggests that CCC

vlas translocated f rom the root zone , vrhere it was applied , to the leaves.

According to Bireclca (1967), a majority of radioactive CCC supplied to the

root of wheat was translocaLed to the leaves and a sinii-lar observation hras

made by Dekhuì-jzen and Vonk (1974). However, the marked decrease in the

level of CCC under hrater stress, as in Table III.3.1, is ha::d to explain though

the possibility ttrat CCC night have been degradecl fasLer under water stress

is not over-ruled.

The apparent lack of CCC in prornoting the accumulation of solut-es

'under l^/atel: stress may explain the lack of osmotic adjustment as discussed

in Section III .3.4.

BO

TII.5 EFF]ICT OF CCC ON i,BA}- DIFFUSIVE RESIS'IAI''ICE

III.5.1 Introduction

It has been reported that CCC induces stomatal closure and,

thereby, increases leaf diffusive resj-stance. l'{ishra and Pradhan (1968

and. 1972) reportecl that CCC treatment caused stornatal closure in tomato.

Pill et a1. Q979) observed an increased leaf diffusive resistance in tomato

due to CCC treatment and similar observations have been made with sunflower

by Loverr and campbell (1973) ancl ruheat by De et a7. (1982).

Since CCC-induced stomatal closure would inply reduced trans-

pirational water loss, it became imperative to study some of the effects of

CCC on Lomato leaf diffusive resistance. Iä thi-s section Lhe effects of CCC

on the diffusive resistance in well-r*¡atered tomato plants is exani-ned.

Firstly, the effect of CCC on the diurnal pattern of leaf

diffusive resistance was stuclied in the glasshouse as well as in the controlled-

environment growth room. Three and a half week o1d tomato plants groln in

the glasshouse, were treated rvith 1OO0 pprn CCC as a soil drench and the

control plants were hratered as normal. 0n the 4th day after CCC treatment '

leaf diffusive resistances were measured with a Li-Cor diffusive resistance

pororneter, as outlined in Section IT.8.1.2, on both the adaxial (upper) and

abaxial (lorver) sttrfaces of the uppermost fu1ly developed leaf (4th leaf)'

The measurements \,/ere done over a day-length period of 10 hours - frorn B a'm'

to 6 p.m. In the controllecl--environment gro\dth room experiment, however,

the adaxial diffusive resistance was also monitored daily at about 9.30 a.n'

over a periocl of seven days, starLing from the first day after CCC treatment.

Diffusive resistance measurelnents were replicated 3 times. In another

experiment, about 4 weeks o1d plants were treated with 1000 ppm CCC' Five

days after CCC treatnent, the adaxial and abaxial diffusive resistances of

the 4th leaf, leaf water potential, transpiration rate - in intact plant and

excised leaves, and leaf area were neasured. At the samé tinle, in anotherbatch

of plant-g after neasuring the adaxial and abaxial leaf diffusive resistances of

the 4th leaves, they were excised and the transpiration rates of either the

adaxial or abaxial surface measured by covering one surface with vacuum

grease to block transpirationa-l- vrater loss from that surface. !-ifteen days

after CCC treatmcnt, the adaxial ancl abaxial dj-ffusive resistances, leaf

water potential, transpiration rate and leaf area were measurecl on the last

B]

batch of pJ-anLs . This experirnent. rr'as car l. ied out in the grorvttt roout atld

replicated three tines"

III.5,2 Results

The results of a comparative study of the diurnal pattern of leaf

diffusj-ve ::esj-stance in CCC-treated and untreated plants in the glasshouse

and controlled-environment growth room are presented in t'igs. III.5.1 and

III.5.2. In the glasshouse the adaxial resistances of both the CCC-treated

ancl untreatecl plants shorved a slight decrease in the afLernoon (i"u. 12-16 hr)

and then increased (Fig. III.5.1.). In the growth room, t,hese adaxial re-

sisLances showed an increasing cliurnal pattern (Þ-ig. III .5.2a). Abaxial

resistances (both CCC and non-CCC) did not shorv any tnarked diurnal- variatj'on

(Figs. III.5. Lb and III.5.2b) under the trvo growth envirounent--s. CCC

markedly increased adaxial diffusive resistance irrespective of the growth

environment but did not influence the abaxial resistance (Figs. III.5.1 and

III.5.2). This CCC-induced increase in adaxial resistance comnenced a day

after the CCC treatrìent and persistecl for 7 days, though after the 4th day

the increase slightly dirninished (Fig. III.5.3).

A further aim was to explore the growth retardation effect of CCC

and its relaLionship with the CCC-induced j-ncrease in leaf diffusive resist-ance. Table III.5.1 shows that 5 days after CCC treatment, when the growth

of plant.s has not been retarded (plant height and leaf area are not reduced),

it caused an increase in the adaxial diffusive resistance, but not the

abaxial , which i{as attendecl with a decrease j.n Lranspiration rate. Fifteen

days after CCC application, the height of the plant and the leaf area were

signifì-cantly reduced by CCC which did not cause a significant increase in

adaxial resistance nor a significant reduction in transpiration rate. This

suggests that the CCC-induced increase in adaxial resj-stance and, hence,

decreased transpiration may diminlsh.rvhen CCC treatment c.auses a reduction

in leaf area (or growth). CCC may also be metabolised by this time, or be

translocated elsewhere in the plant.

The relationship betrqeen diffusive resistauc.e and transpirationby adaxial ancl abaxial surfaces in light and j-n darkness ì.s presented in

Table ]]tI.5.2. CCC increaserl the aclaxial djffusive resistance in light and

hence decreasecl the transpirational water loss from tlre adaxial surface r'¡ith--

FrG. III . 5. 1 :

B2

Diurnal pat-.tern of (a) adaxial and

(b) abaxial diffusive resistancesin CCC-treated and conLrol plants,in the glasshouse.

@ ) ControlA) CCC

((

Aba

xial

Diff

usiv

e

Res

ista

nce(

sec

cm-l)

Þæ 6

Ada

xial

Diff

usiv

e R

esis

tanc

e(se

c cm

-l)

à@

l\) o

g)

o

oo>

>>

t>

o

o)¡\

)

I@ _L o

fJ = oË o g 9) i-,

o

-¡ o) æ

'Ê 4" I t

æ o

I 3i o f\)

o o ß) i..,

o æ

ot I

ol>

FIG. I]I.5.2:

ocOJ

Diurnal pattern of (a) adaxial and

(b) abaxjal diffusive resistancesin CCC-treated and conLrol plants,in the grorvth r:oonì.

ControlCCC

((

eA

))

clI

EC)

ooU'

oC)c(ú

.U'g,oEoU'5:=o(ú.T(úE

-lEoC)oU)

oocG

.9U)o(E

o.ìU)

Eoñ't(U

-o

a1ó

t2^

'

o

A

orf d¡ I

A

Â,

q,I

Oo

ró t8

AA

oó

o ^'

ô^

4

O¡

o4o

A

o^ê

B

44

^^

o

oo

Itooo

r0 12 14

Time of Day(Hr.)

oo

I

8b

O¡

B lo 12 t4 ró lB

Time of Day (Hr.)

p

FrG. rIr.5.3:

B4

Effect of CCC on adaxial diffusiveresistance rvithin a period of7 days.

ControlCCC

@A

((

))

Ada

xial

Diff

usiv

e R

esis

tanc

e (s

ec c

r#)

J o.J lu

o

oN

Ào)

æ

a

O

oÞ

o>

I o Þ rfì o a o o o Þ tt tt o 0) r+ o ¿ a. 0) @

J N (r) Þ ('l o)

O

\¡o>

TABLE III.5.1: Relationship beEween plant size, diffusive resistance ard Eranspiration.

-5da after CCC treatment

\,{ater Plantpotential height(-bars) (ct)

Leafarea

Diffusive re(sec cm

Adaxial

sistance-1)Abaxial

Transoiration raÈe

G'/anz/nr.)Intact plant Excised leaf(¿'2)

6.32 10.68 J.JJ

L.S.D.5% - 2.46 0.08 0'0b

(b) - 15 days afLer CCC treatment

0.45 0.35

Control

CCC

3.67

3.67

2r.87

i9 .60

6.37

T2.T3

8.9s

7 .66

11 .08

12.o4

2.66

3.06

3.16

0.64

0.56

o.4?.

0.57

nÀq

0.39

oo(¡

Control

ccc

3 .91 40.90

22.r73 .83

L.S.D.5% 5.80 2.80

TABLE III.5 2z

B6

Diffusj-ve resistance of intact plant and transpiration rate ofthe Adaxial and Abaxial surfaces of excised leaves 'Measurements were taken 5 days after CCC treatment '

Diffusive resistance (Sec .t-l) TranspiLì-gh

ration rate G/An2 /ttr .)t DarknessLieht Darkness

Adaxial Abaxial Adaxial Abaxial Adaxial Abaxial Adaxial Abaxi-al

7.76 3.96 19.80 9.74 0.30 0.45 0.r9

0. 19 0.30

''qguControl

ccc 10. 89 3. 98 L9 ,29 9 .34 0 .23 0.45

L.S.D. s% 2.69 2.69 0.04

B7

out ary effect on the abaxj-al- surface.

have any effect on adaxj-al and abaxlal

piration rates.

Ti'r darlcness, however , CCC did not

diffusi ve resistauces or their tralls-

1fi.j

¡

III.5.3 Discussion

The slight decrease in adaxial diffusive resista¡Ìces in both the

CCC-treated and non-CCC-treated plants in the afternoon, as happened in the

glasshouse (Fig. III.5.1a), portrays the normal diurnal trend under natur:a1

ligl-rt (Jordan and Ritchie, T97L; Meyer and Green, 19Bl), and this is usually

attributed to fluctuations in the intensity of sunlight" Iä the growlh

room, the increasing diurnal- pattern of adaxial resistance may be attributed

to the effect of the constant light intensity (Sec.tion II.2).

The glasshouse neasurenents of leaf diffusive resistances were

consistent with the growth room measur:enents in that CCC induced an increase

in the adaxial resistance buL not the abaxial resistance under both environ-

ments. This differential effect of CCC in increasing the adaxlal resistance

but not the abaxial can be explained in terms of the different and inde-

pendent behavj.our of the stornata on the two surfaces. Kanemasu and Tanner

(1969b) reported that the aclaxial and abaxial stomata reacted differently to

light. They observed that the abaxial stomata were ful1y open leading to a

low abaxial diffusive resistance at a light intensity which r,¡as lorv enough

Lo close the adaxial stomata and increase adaxial diffusive resisLance. The

same authors (1969a) also found a differential effect of water deficit on

the adaxial and abaxial surface resistances in that the adaxial diffusive

resistance increased sharply at a lower water potential Lhan the adaxial.

Distinct differential behaviour of adaxial and abaxial stomatal diffusive

re:;istances at the same light level has also been found in sorghum and tobacco

(Turner,1968), and in cotton, snap bean, rough lenon, and corn (Erhler and

van Bavel, 1968). Differences between the responses of adaxial and abaxial

stomatal aperture to light in Stachytarpheta indica, Coreopsis grandifTora

and CrotoLaria retusa have been reported by Pemadasa (1979). Apparently , '

therefore, this differential behaviour of the stomaLa in the adaxial and

abaxial leaf surfaces is a widespread phenomenon. The effect of CCC in

differentially increasing adaxial but not abaxial resistance as reported here,

is supported by the work of l4oreshet (1975), who observed that phenyl-mercuric

acetate (PMA), applied to both the adaxj,al and abaxial surfaces of sunflot"er

BB

leaves causecl a Ereater closure of the adaxial stornata than the abaxial'

The CCC-induced inci:ease in adaxial diffusir¡e resistance resulLs

in a decrease in transpiration frorn Lhe adaxial surface, ¿rs evident from

Table III.5.2, and contributes significantly to the reduced tv¿Éerloss fron

the treated planLs (Table III.5.1a). Though the iucreased adaxial re-

sistance due to CCC persists for at least a weelc (Fig. III.5.3), the effect

decreases in about 2 weeks (Table III.5.lb). This agrees r'¡ith evidence

from Mishra and Pradhan (Lg72), who observed a decrease in the magnitude of

CCC-índuced stomatal closure in tomato with time.

III.6 EFFECTS OF CCC AND \¡¡ATER STRESS ON LEAF DIIIFUSTVB RESISTANCE

III.6.1 InLroduction

The effect of CCC on leaf diffusive resistancg as discussecl inSection III.5, indicated that CCC treatment caused a differential increase

in adaxial diffusive resistance but not in abaxial, in well-watered tornato

plants. Since CCC treatment induces the maintenance of higher water

potential.in the treated plants, as has been mentioned previously in ttristhesis, it became necessary to investigate the effects of CCC and water stress

on leaf diffusive resistance.

The materials and methods used v¡ere the same as described in

Sectíon III.3.3.1 and daEa preænted below involving leaf diffusive resistance

were obtained by measurements on the same planls as in Section III.3.3.1.Leaf diffusive resistance was measured with a Li-Cor diffusive resistance

porometer and measurements were made between 8.30 a.m. and 10.00 a.m.

III.6.2 Results

The data for water potential has already been presented inFig. III.3.4 (Section III .3.3.2). The relaLior-rships betr+een"adaxial and

abaxial diffusive resistances, and the time of stress and recovery from stress,

are presented in Figs. III.6.1 and IIf.6.2, respectively. CCC delayed the

sharp increase in both the adaxial and abaxial diffusive resistances and the

diffusive resistances of the CCC-treatecl plants remaj-ned significantly lower

}{I

I

r

((

((

)).,i

t[T

FIG. III.6.1:

ConLrol -

ccc

B9

Effect of CCC on adaxial cliffusiveresistance under stress and recovery.

) F.C.) Stress

F. C.Stress

eo

AA

Ì

I

á-'EC)

oC).n

ooc(ú

.9U,ocro.zØ

Eo(ü'n(úE

+o

30

20

lo

0

L.s.D. 5% Ï

\It

tew at ered

\^

¡--O--¡

\\

I

I

ilÌll'sJ

42o I

Time of Stress and Recovery (daYs)

I

FIG. TII.6.2:

Control-

ccc

90

Effect of CCC on abaxial diffusiveresistance under stress and recovery.

F.C.Stress

F.C.Stress

co

A

^

((

((

))

))

I

j

i

I

I

I

1¡1boooU'

ooc(ú

.9U'ocÍ.o.lU'

=oCÚ't(ú-o

40

30

20

l0

L.S.D. 5% r

4

ll

llr

lt

II

tRewatered g

Lb

--.--

o

ô-ô

2o I

Time of stress and RecoverY (daYs)

1

91

than those of the untreatecl plant-s til1 the 5t-h day of stress on the aclaxial

surface, and even longer on ttre abaxial surface. \{hen planLs uncler stress

were re\{ater:ed , adaxial resistances did tlot l:ecovel: to normal values af ter a

day and was higher j,n Lhe CCC trcatment; abaxi-a1 resistances recovered to

normal values and did not show any CCC effect. Again, in the well-watered

plants, CCC induced an increase in adaxial resistance (Flg. III.6.1) but

not abaxial.

T¡e relationships between adaxial ¿rnd abaxial resistatlces ancl

water potential showed a linear scatter but di d not shor+ any CCC-mediated

effect on either the adaxial nor abaxial resistances (Figs. III.6.3 and

III.6.4). iühen diffusive resistance values were transformed into thei-r

reciprocals, which are normally referred to as conductances, the scatters

depicted curves, shown in Figs. III.6.5 and TII.6.6 for adaxial and abaxial,

respectively, and there \{as no effec.t of CCC. tsoth the adaxial and abaxial

conductances decreaserl a s hrater potential decl-ined but became fairlyconsLant below -7 bars and -9 bars in the adaxial and abaxial, respectj-vely.

III.6.3 Discussion

The finding that adaxial and abaxial diffusive resistauces are

initally lower in the CCC-treated plants under water stress may imply that

the stomata on both surfaces do not close as quickly as in the untreated

plants under stress. This effect may be explaÍ-ned in terms of the CCC treat-rnent delaying the onset of severe plant vlater deficit in the t.reated plants,

as shown in Fig. III.3.4, in response to soil water deficit. As eviclent

from Figs. III.6.5 and III.6.6, CCC has no effect on the decline of leaf

conductance with declining l/ater potential; however, there appears to be a

threshold waLer potential of about -7 bars and -9 bars for adaxial and

abaxial stomatal closure, respectively. Hsiao (1973) has discussed the

existence of a threshold water potential, below which leaf diffusive resistance

and, therefore, stomaLal opening, renains coustant. The present results

agree with the fincling of Duniway (I97I) in that the water potential threshold

for stonatal closure in tomato is about -7 and -9 bars. lloreover, the fact

that abaxial stomata cl-ose at a Lov¡er: watel: potential than adaxial stomala

has already been found 1n snap bean by Kanernastt and Tanner (1969) and in

cotton by Bror.rt-r et a7. (1.976).

FIG rrr.6.3:

92

Relationship belween water potentialand adaxial diffusive resistance.

F.C.Stress

F.C.SLress

Control -

CCC

@

o

A

^

))

((

))

((

oA

o

TEoooÎn

ooc(ü

.9(rtoîEo.àU'J:EoEx(úE

40

30

20

l0

o

o

12 14 ló

AA

A

AAAA

^'

O

ooaoot

024 6 I l0Water Potential(-bars)

FIG. II-1.6.4:

Control -

CCC

93

Relationship between water potentialand abaxial diffusive resisLance.

F.C.Stress

F.C.Stress

@

o

Á.

^

((

((

))

))

Ao

A

oo

Ltooou,ooc(ú

.9ÎDq)frorn5:=o.Exõ

T¡

40

30

20

l0

o

12 14 ló

A

^tr^t

lr|'o2 4 6 I l0

Water potential (-Bars)

FIG. III 6

Control -

ccc

5

94

Relationship between water potentialand adaxial conducLance.

F.C.SLress

F.C.Stress

@

o

A

^

((

((

))

))

ít(,og,

EooC)C(ft

ofEcooEx(úE

o.4

o.2

o

o

^A^^

oo

o'

{r

o

a

024

AAo OA

6 I toWater Potential(-Bars)

12 14 tó

o AO

FIG. III.6.6:

Control -

ccc

9s

Relationship between hrater potentialand abaxial conductance.

F.C.Stress

F.C.Stress

€o

À

^

))

((

))

((

0.8

o.4

o.2

oo

Ao

^.

o'ó ^

Iooa,

EoooE(ú

oãocooEx(ú.c¡

I

A

A

o o A oao

024 6 I l0

Water Potential (-Bars)

12 14 ló

96

I]I.7 EF]I]iCTS OF CCC AND \^IATEìì STRESS 0N 'IOTAL WATER I-0SS

III.7.1 Introduction

Results presented so far inclicate that the CCC-induced maintenallce

of r,¡ater statrls under stress may be explained in terms of the initial reducecl

transpirational water loss in CCC-treated planLs, which l.earls to a delay 1-n

the onset of i-nternal water stress in the treated pl.anLs. It has already

been found that CCC*treated pl.ants use water rìore economicalÌy under drought

(El Damary et a7., 1965; Farah, 1969; Plaut et a7.,1964). A stud¡'was

theref ore conducted to explore the ef f ects of CCC on total \!'ater loss.

The tomato plants were grown j-n 1 kg recycled soj 1 (l{aite

Institute) 1,n I27 mm (5 ins) pols. Hoagland's solution was supplied as a

supplementation whenever required. CCC 1000 ppm was applied as soil drench

at 150 m1/pot to 3å v¡eeks o1d plants. \rlater stress was imposed by with-

holdíng water from the plants, 4 days after CCC treatment. All the pots

were embedcled in polyLhene bags to minimize water loss from the soil. Pot

weights were recordecl daily at the start of the 1 ight period in the grolt-h

room (i.e. 6.00 a.m.) and at the end of the tight period (10.00 P.rn.). The

non-stressed plants were returned daily to field capaciLy after vreighing the

pots in the morni-ng. Leaf r'¡ater poLential was monitored wiLh the Pressure

bomb during the course of the stress and the experirnent was replicated three

times.

III.7.2 Results

The water loss per day in light and in darkness is presented inFÍg. III.7.1. Under well-watered conditions, the water loss during the

light period was significantly less in the CCC-treated plants than the un-

treated planLs, but there v/as no such difference in the dark period.

By the 3rd day of stress the water loss per day during the light period, inthe non-CCC-treated plants, had decreased dramatically such that it was less

than in the CCC-treated plants til1 the 5th day of stress.

CCC significantly reduced the cumulative water loss in the well-watered plants. Under stress, however, the cunulative watetî loss was signi-ficantly decreased on the 3rd and 4th days of stress in the non-CCC-treated

and CCC-treated plants respectively (Iig " III .7 .2) .

}-IG. IIT.7.1:

Water loss duringthe light period

liater loss duringthe dark period

91

Effects of CCC and rvater stresson water loss/day.

I{ell-watered plantsSLressed plants

ControlCCC

) Control) ccc

(u)(b)

@

A

o

^

))

((

((

G60(\toi40o

f;zo3

a

b

f t.s.o. sy"

It.s.o. sy"

o 12345Time After With-holding Water(Days)

67

1234567Time After With-holding Water(Days)

60'o,

(úooCI'o

o(ú

=

80

40

20

o

FIG. I'II.7.22

ccc

(6)(o)

Control -

9B

Effects of CCC and water stresson curnulative water loss.

F.C.Stress

F. C.Stress

A

^

((

))

o)cf,(t,oJ¡-o(ú

=o.=(ú

=E5o

640

560

480

400

320

240

L.S.D. 5% I

'v-¿é4-À--

-Å=--=-:E -o-^

160

BO

123456Time After With-holding Water(Days)

7

99

The relationship between water loss/day and water potentialpresented in !'ig. IIT.7"3 shows that the water loss/day decreased as \,"'ater

potential declined in response to soil water deficit, til1 about -B bars

when the rate slowed dorvn. Cumulative water 1oss, however, increased withdeclining water potential until about -B bars when the rate slowed dorvn,

as sholn in Fig. ITI .7.4.

III.7.3 Discussion

The decrease in the water loss due to CCC treatment under normal

well-watered conditions may be attributable to the CCC-induced increase inleaf diffusive resistancg largely on the adaxial surface, as discussed inSection III.5. I'lhen the plants were subjected to soil vrater depletion, the

CCC-treated plants, by virtue of their reduced water loss initially, de1-a1.sfl

the onset of severe plant water deficit and maintained better water status

and, therefore, their stomata remained more open than in the untreated plants,

as in Figs. III.6.1 and III.6.2. This is reflected in the non-CCC-treated.

plants controlling further loss of water earlier (3rd day of stress) than the

CCC-treated plants (4th day of stress) as shown in Fig. III.7.I.

The amount d water required to be transpired for the water potentialto drop to a given 1eve1 ras unaffected by CCC. A threshold wat.er potentialof -8 bars was found for both CCC-treated and untreaLed plants under stressand this is consistent with the threshold water potential of -7 bars to -9 bars

for stomatal closure as discussed in Section fII.6.3.

ÏlÍij

FIG. IIT.7.3:

r00

Relationship beLween water potentialand rvater loss/day.

ControlCCC

øÂ

((

))

t

i

III

i

þ

4l

iti

70

60

50

40

30

20

10

o

I

lr

A

o

^.

o)

(úoCI'g,oJ

oG,

=NI

'ôI

024

o

^.

ao

6 I 10 12

Water Potential(-bars)

io

14

i

iI

t

I!,

TI

I

t

i

WA,l'ti: lr'l5TiTÌJIt

tIt{RARY,

FIG. III.I .42

101

Relat1onship betvreen water potentialand cumulative \,/ater loss.

ControlCCC

@

^

((

))-tti

þ

400

300

200

100

aa

aA

12 14

AA

a

ct¡(r,CI'o

o(ü

=o.ì(ú

E5o

a

A

46810Water Potential(-bars)

)

II

I

ri

iirl

'i

IT

I

t,

o

^

02

I

II

ro2

CI.IAPTER IV

GENERAL DISCUSS]ON

It is quite a \^rídespread phenornenon that CCC increases the

abitity of sorne plants to resisL drought, and various mechanislns have been

proposed to explain this CCC-induced drought resisLance.

Some workers claim that CCC increases the ability of plants

to endure or tolerate st-ress through CCC-mediated netabolic changes suclt

that even at low tissue t,'trter potent1al, CCC-treated plants rnay retaj-n

t-heir turgor possibly tl-rrough CCC-mediated accumulation of solutes.

Singh et a7. (1973), reported a pronotion of proline accumulatjon in CCC-

treated wheat plants subjected to an osmotic shock and suggested thal the

CCC-induced accumulated pro1ine may enhance recovery from water stress.

Stoddart (1.964) observed that osrnotic substances like polysaccharides and

amino acids accumulate in the presence of CCC and proposed the possible

involvement of these solutes in the CCC-inducecl resistance to drought.

Marth and Frank (f961) speculated that the increased tolerance of CCC-

treated soybean plants to soluble salts may involve some CCC-induced

meLabolic change since the populaLion of mites feeding on the CCC-treated

plants was reduced.

Other workers explain the CCC-induced resistance to drought

in terms of reduced transpiration and increased root/shoot ratio, such

that under water stress the CCC-treated plants have favourable waLer status.

Robertson and Greenway (1973) reported that CCC-treated maize and wheat

plants avoided drought by delaying the onset of severe internal water

deficit, by virtue of reduced leaf area and increased root/shoot ratio.PlauL et a7. (1964), claimed that a decrease in transpiration rate and

top/root ratio in CCC-treated bean plants conLributed to their prolonged

survival under water stress. Moreover CCC-induced closure of stomata has

been reported in tonato (l'lishra and Pradhan, 1967; 1972; P|II et a7-

(1973), cowpea (Tmbamba, I972), sunflower (Lovett and carnbell, 1973), attd

wleat (De et a1. r I9B2) and this has been associated with reduccd trans-pirational water loss and, hence, better rvater status under water stress.

103

Thus, CCC-induced drought resist-ance involves nleLabol-ic,

morphological ancl physiologj-cal changes due to the CCC treatnent; horvever,

there has not been any detailed study of horv these changes affect the water

relations under sLress. In the present work much emphasis r'¡as placed on

mechanisms involved in the CCC-induced ilrought resistance of totlato '

independent of the CCC effect on grorvth.

Results from this çe¡|ç Tevealedthat CCC, at a concentration of

10OO ppm, enâbled the treated tornato plants to delay wilting ancl survive

longer: under water stress even ivhen growth rr¡as not rel-arded (irig. III.1'2)'

At this concentration, however, CCC exterted an inhibitory effect on the

height of the plants 6 days after its application (Fig. III.1.3). \n/ittr*'er

and Tolbert (1960) and van Bragt (1969) also found that CCC retarded the

height of tomato 5-7 days after i-ts application. The reduction in height

was followed by a reduction in total leaf area resulting from the reduction

in Lhe area of the leaves formed or sti1l developing after CCC application

(Fie. III.1.4). This CCC-inhibition of the growth of developing leaf

tissues can be explainêd by the findings of Sachs et a7. (f960) and Russel

and Kimmins (L912), that the retardation of growth by CCC might be attri-

buted to the inhibition of meristematic activity and cel1 elongation.

Once the stature of the plant was reduced, the dry weight of the shoot was

also recluced, but not the root, rvhich resulted in an increase in the rootf

shoot ratio (Tables III.1.2 and III.1.3).

\rlhether CCC retarded grorvth or not, under,water stress induced

by with-holding waLer, CCC-treated plants maintained higher water potential

and this better plant water status might have accounted for the prolonged

survival under water stress, though CCC did not sustain growth.

lrthen ccc has not affected gro\^/th the ccc-induced ::esistance to

water stress could not be explained in terms of CCC-induced osmotic adjust-

ment since CCC-treated plants clid not lower their osmotic potential enough

(Figs. III.3.5 and III.3.6) to maintain turgor at 1ow tissue \^/ater poLential.

According to luforgan (Ig77b) and Turner (1974), in plants than can adjust

osmotically to water stress, a unit decrease in water potenti'al results

in such great lowering of osmotic potential that turgor potential is

maintained at lower Lissue water potential. Jn addition, CCC did not alter

104

the rnoisture release curve (Fig. III.3.3) and, accordíng to Jones ancl

Turner (1978), osmotic adjustment may involve shifting the moisture release

curve such that the decrease in R\{C is less rvith a unit decrease in tsater

potential. Furthermore, the apparent lack of ccc to s-ignificantly

increase the accumulation of proli-ne (Figs. III"4.2 and III'4'4) or any

other quaternary amrnonium conpound (Figs. III.4.5 and III'4'6 and Tabl-e

III.4.1) may suggest their non-involvement in the maintenance of better

water status under stress bY CCC.

The other evidence from this thesis showing a differentialincrease in adaxial diffusive resistance but not abaxial (Figs. III'5'1

and III.5.2), and the rapid increase in adaxial diffusive resisLance due

to CCC without an effectongrowth (Fig. III.5.3) could be interpreted as

CCC inducing stomatal closure in the adaxial leaf surface. The possì bility

of CCC inducing stomatal closure of the adaxial surface but not the

abaxial has been discussed in Section III.5.3. This CCC-i-nduced closure

of adaxial stomata in normal well-watered tomato plants rvas consistent

with the reduced transplrational water loss from the adaxial surface

(Table III.5.2) ancl this, possibly, accounted for the reduced water loss in

the CCC-treated plants (Fig. III.7.1a) under field capacity. trtlater stress

also caused stomatal closure on both the adaxial and abaxial surfaces but

this response was quicker in the non-.CCC-treated plants (Irigs. II1.6.1 and

III.6.2). Since the water potential thresholds for stomatal closure (on

both adaxial and abaxial surfaces) r{ere consistent rvith the water potential

thresholds for controlling excessive water loss under stress (Figs. III.6.5,III.6.6, III.7.3 and III.7.4), and were unaffected by CCC, it may be argued

that CCC delayed the time at which the water poLential threshold for stomatal

closure was reached; this agrees with the initial l-ess rapid decline inr{rater potential in the CCC-treated plants under water stress.

llhen CCC has affected gr:owth the CCC-induced adaxial djffusiveresistance may diminish (Table III.5.lb) but the reduced leaf area will in-evitably reduce water loss and maintain higher water potential.

In conclusion, when gror'¡th is unaf f ected by CCC Lreat-ment , the

closure of the adaxial sLomata results in reducei water loss under normal

well-watered conclitions. This initial control of water loss enables the

CCC-treated plants to rnaintain higher tissue water potential, thus delaying

105

the onset of severe v/aLer deficiL in Lhe plants and prolonging theirsurvival under water stress. This CCC-induced prolonged survival under

v,ra¡er stress did not involve sufficient osmotic adjustment to permit the

CCC-treated plants to sustain growth under low tisstle h'ater potential.This could be due to the lack of CCC-induced metabolic changes under water

stress. However, when CCC has retarded growth, the increased root/shoot

ratio and reduced leaf area will inevitably play a very significant role in

the CCC-induced resisLance to water sLress.

106

BIBLiOGRAPTIY

. and VERKERK, K. (1970). Grorvth, f lowering and f rui,tingin tomatoes in relation to temperattlre, cycocel and G'A'Neth. J. agric. Sci. 18: 105-110.

CANIIAI,{, A.E. and IIARRIS, G.P. (1973). llff ect's of CCC on theformation and abortion of flowers in the first influolesc-ellceof tomato (Lycopersicon escul-ertun l"iill. ) .Ann. Bot. 422 611-625,

ABDALLA, A"A

AIJDUL, K . S

ADEDIPE,

ADEDIPE,

ACEVEDO, E. , HSAIO, T.C. and IIENDERSON, D.W. (1971). Irnnediate and subse-quent gr:ortth responses of maize leaves to changes in waLer

status. P7a:'t Physiol . 48; 63I-636,

N.O. and OR},IROD, D.P. (1970). Plant gror,rth retardants and

phosphorus metabolism. Ibid. 2I: 4I4-4I1.

N.O. and ORMROD, D.P . (1972.). Vegetative grorvth r:esponse of pea

plants to ccc and phosfon in relaLion to phosphorus nutrition.J. exp. Bot. 232 842-848.

N.0., ORMROD, D.P. and MAURER, A.R. (1968). Response of pea

piants to soil and foliar applications of cycocel (2-chloroethyl)trimerhylammonj.urn chlorjde. Can. J. P7. Sci. 48: 323-325,

N.0., ORMROD, D.P. and Ì,4AURER, A.R. (1969). The response of pea

piants to low concentrations of Cycocel, Phosfon ancl B-Nine.J. Aner, Soc. hort. Sci. 942 32I-3.

ADEDIPE,

ADEDTPE

ASIIER, W.C. (1963). Effects of 2-chloroethyltrimethylamrnonium chlorideand 2'r4-dichlorobenzyltributylphosphonium chloride on growth

and transpiration of slash pi-ne. Nature 20O-: 9I2'

ASPINALL, D. and PALEG, L.G. (1981). Proline accutnulation : Physiologicalaspects. Int ,The PhysioTogy and Biochenistry of DrouglttResistance in PTants'. Eds. Paleg, L.G. and Aspinall, D.

Academic Press, Australia. pp. 205-259 '

AUGUSTIN, A.M., DUYSBN, M.E. and \,üILKINSON, G.E. (f968). Internal tvaterbalance of barley under soil moisture sLress 'Plant PhYsiol. 432 968-972.

BARNES, M.F., LIGHT, E.N. and LANG, A. (1969). The action of plant groylh --retarrlants on terpenoid biosynthesis. PTanta (Ber7.) Ët L72-L82.

BARRS, Il.D. and WEATHERLY, P.E. (1962). A re-examinatj.on of the relativeturgidity technlque for estimating water deficits in leavesAust. J. Bio7" Sci. 15: 413-428.

BARRS, H.D. (f968). Determination of water deficits in plant tissues.In: 'l,later Deficits anc| PTant Grc¡wth'. Vol.1. Ed. Kozlowski, T'T¡ - - r^--i - D--^^^ Àì^., v^-1, ^-'l T ^-.lnn ññ )?6-?68-¡lLdugrllru IIEùÐ' ¡ruYY ' Yr

107

BEGG, J ìi. ancl TUIìNER, N. C. (1970) .

tobacco . PTant PhYsiel.\,later potential gradients in field46: 343-346.

Acìaptation to water clef ic j ts 'BEGG, J ll. and TURNER, N.C" (1976).Adv. Agron. 292 16I-217'

BENGSTON, C., KLOCK^RE,8., KLOCKARE, R., LARSSON' S. and SUNDQUIST', C' (1978)'

The after-effect of water stress on chlorophyll formation during

Ereeninganc]thelevelsofabsciclcaciclandprolineindarkgrown wÉeau seedlings. physioT. PTant /+32 2O5-2I2.

BHATTACHARJEE, S.K", DAS GUPTA, K. and B0SB, T.K" (1971)' B-nine', an

effect-j-vegrowthreLardantottDahlj-a;ineffectivenessofCCC.QYton 2Bz 61-65.

BIRECKA, I{. ar,¿ åEgnO\^lSKI, Z. (1966). Influence of (2-chloroethvl)trimãtnylun',ro,,iut .t-,iotj ¿" (CCC) on photosynthetic activity ancl

frostresistanceoftonlatoplants.Bull.Acad.Pol.Sci.Cl.V.'Ser. Sci. Bio7. L4: 361-313'

BIRBCKA, H. (1967). Translocation and distribution of 11tc-lubelled' (Z-.hioroethyl) tri-methylamrnonium (CCC) in wheat'

8u11. Acad. Þo1. Sci. Ci. V., Ser. Sci. Bio7. 15: 701-714,

BLTNN, R.C. (1967). PlanEgrowLh r:egu1,ant. Biochemical behaviour of2-chloroethyl tiimethylamrnonium chlorj,de j n rvheat and in rats 'J, Agr. Food Chen' 15" 984-9BB'

BLUI'{, A. (1975). Effect of the Bp gene on epicuticular wax and the water

relaLions of Sorghun bicoTor L' (lloench) 'IsraeT J. Bot. 242 50-51'

BOYBR, J"S. (1969). lleasurement of the \^/ater status of plants'Ann. Rev. P7. PhysioT' 2Oz 351-364'

BOYER, J.S. (1970). Leaf enlargement and metabolic rates in corn' soybean'

and sunfl-ower at various leaf water potentlal 'Pl.ant PhYsioT. 462 233-235'

BOYBR, J.S . (1971). Resistances to \^Iater transport in soybean' beatr and

sunflower . CroP Sci. 11: 4O3-4O7 '

. (Lgl4). Water transport in plants : mechanisms of apparent

changes in resistance during absorption. PTanta I1-7: IB7-2O7 '

. ) JORDAN, \^/.R. and THOI'ÍAS, J'C ' (1976)' \riater sLress lnduced

alterations of the stomatal response to rlecreases in leaf water

potential. PhysioT. Plant 3-7: 1-5'

M. and STUART, N.W. (1961). Comparative Pial! grot"th acLì-vity

of A110-1618, Phosphon and CCC. Bot' Gaz' I23" 5I-51 '

M. (f964). Physiology of grovrth retarding chemicals'Ann. Rev. PL, PhYsioT. 15: 27I-3O2'

BOYER, J.S

BROl^lN , K.W

CATHEY, ll

CATIIEY, H

108

CA'IHEY, H.!1. (Ig75). Comparative p1.ant gr-orvth-retarcling activities ofancymiáo1 wiLh ACPC, phòsphon, chlorntequat ar-rcl SADII on

ornanental plant species. Hortscience IO: 204-216.

CONNOR, D.J. and TUNSTALL, B.R. (1968). Tissue \{ater rel'ations for brigalowand mu1ga. Aust- J. tsot. 16-: 487-490'

COOMBE, B.G. (f965). Increase in fruit set of Vitis vinifera by treatnentwith growth retardants. Nature 2O5¿ 305-306'

CRITTENDON, C.E. and KIPLINGER, D.C. (f969). Effects of B-9 and Cycocel

on sone factors influencing leaf color and sten lengthchrysanthemum and poinsettla. Ohio F1or. Ass. Bull' No'4Bl "

2-4'

CROSSAN, D.F. and FIELDIIOUSE, D.J. (1964). A comparison of dwarfing and

other conpounds with and without fixed coPl?r fungicide forcontrol of bacterial spot of pepper . Plant Dis. Rep. 4Bz

s49-ss0.

CROSS, B.E. and Ì"IEYERS, P.L. (1969). The ef f ect of plant growth retardantson the biosynthesis of diterpenes by Gibberel-7a fujikuroi.Phytochen. Bz 79-83.

CUTLER, J.M. and RAINS, D.W. (1978). Effects of water stress and hardeningon the inEernal water relations and osmotic const-ituents ofcotton leaves. PhysioT. PTant 422 26I-268'

DAVIES, W.J. (Ig77). Stomatal responses to water stress and light in plantsgrownincontrolledenvirolìmentsandinthefield.Crop Sci. L7: 735-140.

DE, R., GIRI, G., SARAN, G., SINGH, R.K. and CHATURVEDI, G.S. (1982).l"lodiiication of rvater úalance of dryl and rvheat through the use

of chlormequat chloride. J. agric. sci. camb. 98: 593-597'

DEKHUIJZEN, ll.l'{. and VONK, C.R. (T974). The distribution and degradation-of chlormequat in whåat pi-ants. Pest. Biochen, PhysioT' 4" 346-55'

DEVAY, M., SHARAKY, 14., FBHER, M. and KOVACS, I. (1970). some physiolog-ica1effects of CCC on PhaseoTus vuTgaris. Proc. Intern. Hort. Congr'

XVITI. l. No.86, P.46'

DIERCKS, R. (1965). The control of eye-spot disease of tvheaL (CercosporeTla

herpottichoicJes) with chlorocholine chloride'Z. pflanzenkrankh, PfTanzenschutz. l2z 25I-21I; as cited inChen. Absts. 63: 10596d-e.

DIMOND, A.E. (1966). Pressure and flow relations in vascularof the tomato p1ant. PTant Physiol' 4Iz 119-131'

DOUGLAS, T.J. (Ig74). The effects of planL grorvth retardants on

biosynthesis in tobacco seedlings' Ph'D' Thesis'UniversitY of Adelaide. (179P)'

sterolThe

DUNBERG A. and ELIASON, L. (Lg72). Effects of grorvth retardants on Norway

Spruce (Picea abies). PhysioT' Plant 262 302-305'

J.M. ( 1971) . Water relations of Fusariur¡r wilt in tomato 'Physiol. Pl" Path. Iz 531-546"

DUNTWAY,

109

DySON, p.l,/. (1965). EffecLs of gibbereJ 1ic acicl and (2-chl oroetl-ryl)' trr'pethylammonj-um ch1òride on potato grotvth and developnent.Jour . Sci . Foocl ,&, Agric. I Q: 542-549 '

ERIIRET, D.L. and BOYER, J.S. (1919). Potassiun loss from stonatal guard. ce1ls at 1or,,¡ water potentials. J. exp. Bot. 30: 225-234"

EL DAI',IATY, Il., XÜtlN, H. and LINSER, Il. (1964). A prelirni-nary i-nvestigationof increasing salt tolerance of pla[ts by applicati.on of(2-chloroethyl) trimethylammonium chloride'Agrochinica B: 1.29-138.

EL DAÌ"IATY, A.H., ttÜtlN, ll. , LIlisER, It. (1965). Water relations of wheatplants under the influence of (2-cl-rloroethyl) trimethylammoniumchloride (CCC) . Physiol. PTanr l9z 650-657 '

EL-FOULY, M.M. and JUNG, J. Qg6g). Some factors affect the degradatiorr of(2_chloroethyl)trinreth)']amrnonium.chloridebywheatplantextracts, Experientia. 252 587-588.

EL-FOULY, M.M., ISMAIL, A.A. and AIIDALLA, F.E. (1970). Uptake, distributionancl translocation of J'ZP absorbed through roots of cottonseedlings as affected with CCC treatments'Physiol. PTant 232 686-690.

BL-FOULY, M.l'{., BL-HAI'JAIüI, H.4., FA\nlZI, A.F.A. (1971). DifferenL effectsof chlorrnequat on chlorophyll, protein and yield in cotton grown

uncler u.rying water regirnes. Plant and SoiT 352 183-186.

ERHLER, W.L. and VAN BAVEL, C.H.l'\{. (1968). Leaf dif fusion resistance,illuminance and transpiration. PTant PhysioTogy !1t 2OB-2I4.

FARAH, S.M. (1969). Effects of chlorocholine chloride and water regirneon growth, yield and water use of spring wheat 'J . exp. Bot. 2Q'" 658-663.

FELIPPE, G.M. and DALE, J.E. (1968). Effects of a growth.retardant' CCC'

on leaf growth in PhaseoTus vul.garis. PTanta (I3er7.) B0:

328-343.

FISCI{ER, R.A. and SANCHEZ, M. (1979). Drought resistance in spring wheat

cultivars. II. Effects on plant rvater relations'Aust. J. Agric. Res. 30: 801-814.

FORD, C.l4l. and \IIILSON, J.R. (1981). Changes in leve1s of solutes duringosmotic adjustment to water str:ess in leaves of four tropicalpasture spãcies. Aust. J. PTant PhysioT' B: 77-9I'

GARDNER, \^/.R. and EHLTG, C.F. (f965). Physical aspects of the internalwater relations of plant leaves. PTant Physiol. 40: 705-710.

GATES, C.T. (1955). The response of young tomat--o plant to a brief periodof rvater shortage. II. The rvhole plant and its principal parts'Aust. J. Bio7. Sci. B: I9B-2I4.

GAY, A.P. and llURD, R.G. (f975). The inf luence of light on stornaLaldensity in Lhe tomato - New PhytoT. 75-z 37-46'

110

GHOLKB, A.F. and TOLB]]RT, N.E. (1.962). Ef f ect of 2-cirlorcethyl-trirnethylanmoniun chloride on pliosph¿rte absorption and Lrans-location. Pl-ant Physj-s1. U.S.A't-z (Suppl '): xii'

GOWING, D.P. and LEEPER, R.W. (1955). T,nduction of flor+ering in pi.neapple

by $-hydroxyethylhydrazine . Science 722-z 1261 '

IIALEVY, A.H, and KESSLER, B. (1963). . ncreasecl tolerance of bean plantsLo soil drought by rneans of gror+th-retarding substances'Nature IWz 310-311.

HALBVY, A.H. and \^IITT\IJBR, S.H. (1965a). Growth promotigl^in the snapdragon,by CCC, a grorvth retardant ' Naturwiss 522 310"

HALBVY , A. H. and hIITT\,JER, s.I{. ( 1965b ) . Foliar upl-alce and translocation ofrubidium in bean plant-s as affected by root-absorbed growthsubstances. PTant Ph1'5io1' 40: (Supp1 '), xxiv'

HALEVY, A.H. Gg67). Effect of gr:owth retarclants on drought resistance and

long",rity of various flants. Proc. Intern. Hort. Congr. XVII.3: 217-283.

HALEVY, A.H. and SHILO, R. (1970). Promotion of growth and flowering and

increase in content of endogenous gibberellins in GTadioTusplants treated with the growth retardant CCC'

PhysioT. PLant 232 B2O-827.

HALL, A.E. and SCHULZE, E.D. (1980). Drought effects on transpiration and

leaf water status of cowpea in controlled environments.

. Aust. J. P7. PhYsioT. 7z L41-I4l'

HARADA, H. and LANG, A. (1965). Effect of some (2-chloroethyl)trimethylammonium chloride analogues ancl other growth retardantson gibberellin biosynLhesis in Fusariun noniTiforne.PTant Physiol. 40: 176-f83.

HEATHERBELL, D.A. , HOWARD, B.H. and \^/ICKEN, A.J. (1966). The effects ofgrowth retardants on the respiration and coupled phosphory-lationofpreparationsfrometiolatedpeaseedings.Pltytochen. 5z 635-642,

HBIDE, 0.M. (1969). Interaction of growth retardants and Lemperature ingrowtú, flowering regenerãUion, and auxin activity of Begoniax cheimanrha Everett. PhysioT, Pl-ant 222 1001-1012.

IIIRON, R.W.P. and WRIGÌ.IT, S.T.C. (f973). The role of endogenous abscisicacid in the response of plants to stress 'J. Exp. Bot.24z 769-78L.

HOAGLAND, D.R. and ARNON, P.I. (1938). The water culture method for gr:owing

plants without soil. circ. ca7if. agric. Exp. stn.347.

HODGSON, R., PETERSON, W.H. ancl RIKER, A.J. (1949). The Loxicity of poly-saccharides and other large mol-ecules to tonato cuttings'PhytopatltoTogY 392 47-62.

HSAIO, T.C. (1973). Plant responses to waLer stress'Ann. Rev. P7. Physiol. 24t 519-570'

HSAIO, T.C", ACEVEDO, 8., FERERES, E. anrl IIENDERSON, D.hl . (L916). StressrneLabolism : water stress, grot.rtli and osmotic adjttstment.Philos. Trans' R' Soc ' Londàn' (Series B)' 2'l1z 419-5OO'

111

HUI'îPIIRIES,

HUMI'HRIES,

HUMPIIRIES,

HUI'IPHRIBS,

HURD, R.G

E.G. (f963) " Effects ofchloride on Plant growth,Ann , I3ot. ?-l z 5l 7-531 .

TNALL, D. (1970).11 ey. Ann, Bot.

( 2.-chloroethyl ) trimethylantironiumleaf area ' ancl net assímil'ation rate ' '

E.C., \^¡ELBANK, P.J. and WITTS' K'growth and Yield of sPring -wheatÁnn. App7, Bio7. 56-: 351-361"

E.C. (1968). CCC and cereals'

E.C. ancl FRENCH, S.A.\nJ" (1965) ' A.growlh study of sugar. beet

treatecl rvith gibbe'ellic aci¿ and (2-chloroethyl) trimet-hyl-ammoniumchloride(CCC).Ann'App7'Bio7'55:I59-I73'

(1969). Leaf resistance in a glasshouse tonìato crop in relationto leaf position and solar racllation. Netv Phytol. li8: 265-273'

ln/ater stress and34: 393-408.

. (f965). Bffect of CCC on

in the field.

F7d. Crops ¡lbsts. 2L: 9I-99'

apic-a1 morphogenesis

J

HUSAIN, I. and AS

in ba

IMBAI',{BA, s.K , (Lg72). Response of cowpeas. to salinity and (2-chloroethyl)rrimethylammor.riirm c¡tfåti¿" tCCC) , PhysioT. PLant 28z 346-349'

IMBER, D. and TAL, l'1. (1970). Phenotypic reversion of flacca, a wilty*uru'i of Ècimatå by absci.cit acicl . Science 169z 592-593'

JARVIS, P.G. (1975). Water transfer in plants. In z 'Heat ancl Mass Transfer

in the Biosphere. I. ,Iransfer Processes in the PTant Environnent,.Eds. de Vries, D.A'-ått¿ Afgan, N'H' Scripta' Washington' D'C'

PP. 369-394,

JONES, R.L. and PHILLIPS, I.D.J. (1967). Effect of ccc on the gibberellincontent of excisecl sunflower organs.. PTanta (Ber7') JZr 53--59'

JONES, M.M. and TURNER, N.C. (1978). osmotic acljustment in leaves ofsorghum in response to water deficitsl PJant PhysioT' 61: 122-126'

JONES, M.M. and TURNER, N.C. (1980). Osmotic adjustment jn expanding and

fulty"*p',,d"dleavesofsunflowerinresponsetowaterdeficits.- Aust. J. PTant Pltysiol' 7z IBI-I92

JONES, M.M., TURNER, N.C. and OSÌ"IOND, C.B. (1981). Mechanisms of drought

resistance. In:'The PhysioTogyandBiochenistry of Drought

Resistance in p7ants,. Eãs. Paleg, L.G. and Aspinall, D.

Acaclemic Press, Australia ' pP' 15-37 '

JONES, M.M., OSMOND, C.B. and TURNER, N.C. (1980). Accurnulation of solutesin leaves of sorghum and sunflower in response to water''defi-cits'

JORDAN, W.R. and RITCFITE, J"T. (1971). Influence of soil water stress on

evaporation, root uù"otption ancl internal water status of cott"on'

PTant PhYsioT. 4åt 783-7BB'

gro\4/th and develoPment in radish'ih" Uniu"rsity of Adelaide' pp' B2-BB'

Áust. J. PTant Physiol. Tl I93-ZO5'

C. (1980). tr/ater stress andl'laster Agric. Sci. Thesis

'

JOYCE, D

r1.2

JUNTILLA 0.(roso¡.BuddifferentiatiorrinSafjxpertanclraasaffec.tedb;-;h"ioperiod' Lernperal-ure and gr:owth regulators'PhysioT. Plant 49-z 721-l'38 '

KANF,MASU, E.T. ancl T'ANNUR, C+Bi- (l969a) 'snaP beans' i' ftlfIucucc ofPlant PhYsioli t+!? I54l-1552'

Stonatalleaf tva,ter

cli f f usion resi stance ofpotent-ia1

KANEI'îASU, E.T. and TANNEII, C

of snaP beans.1542-1546.

KANEIIÍASU, E.T. , TI.IURTELL, G.lrl. and TANNBR, C.B. (1969) ' Design, calibra-tion and field use of a stonatal diffusion poroneter.Plant PhYsiol" 44¿ BBl-885.

KENDE, H. , NINNEI',IANN, H. , LANG, A. (f 963). Inhibitj-on of gibberellic acidbiosynthesis in Fttsariunt not-tiLifornre by Amo-1618 anrl CCC.

Naturwiss" 50: 599-600.

KHARAi{YAN, N.N, (1967), Bffect of 2-chloroethyltrimethylammonium c}rloride' (CCC) on sorre characteristics of the water conditions in plants 'Soviet PTant PhYsioT. I4: 465-468'

KHARANYAN, N.N. (1969)" Effect of retardant chlorocholine chloride (ccc)

on nìtrogen rnetabolism of plants. Soviet PTant Physiol. 16:

7lg-12r.

KNAVEL, E. (1969). Influence of growth retardants on growth, nutrientcontånt, ard yield of iomato plants grown at various fertilitylevels. J. Aner, Soc. Ilort. Sci ' 942 32-35'

KNIPLING, E.B. (f967). Effect of leaf aging on water deficit - hraterpotentiai relationships of do[wood leaves grorving j n two environ-meuts. PhYsioT. PTant 2Oz 65-72

KOZLOWSKI, T.T. (l968). Inlroductlon. In z ' y/ate:' Def icits ancJ p'Lant grorvth' 'Vof.i. Ed. Koz1orsski, T.T. Academic Press, New York and London'pp.1-19.

KOZLO\^/SKI, T.T . (Lg76) . Water supply and leaf shedding. In z 'WaterDeficits and Plant Grot.ttlt'. Vol.4. Ed. Kozlowski, T.T.Acadenric Press, New York, san Francisco and London. pp. L9l-231'

KRAMER, P.J. (1969). 'P7ant and SoiT Relationships : a [lodern S)'nthesist'McGraw-Hill, New York.

LARTBR, 8.N., SAMII, M. and sosuLSI(T, F.W. (r965). The mor:phological and

pf,y"iofågical effects of CCC on barley, Can. J. P7. Sci. 452

419-427.

LAhTLOR, D.W. (f970). Absorption of polyethylene glycols by_plants and

their åtf.t" on plant growth. New PhytoT" 69¡ 501-513.

LESHAÌ,4, B. (1966). Toxic effects of carbowaxes (polyethylene glycoJs) on

PìnushaTipensisl"Iill.seecllings.PT.SoiT|!2322_324.

LOCKHART, J.A. (1962). Kinetic st.uclies of certain antigibberellíns'PTant PhYsiol. 31-". 159-764'

.8. (f969b). SLomatal djffrrsion resistancerr' Ef fect of 1ight" PJ-ant Phl'5io1' /+4:

MARTH, P.C. and FRANK, J.R. (1961). Increasing tolerance of soybean plantsto sone soluble salts through application of plant groruth re-tardant chenicals. J. Agr" Food Chent, 9¿ 359-361.

l'{AY, L.ll. .and I'{ILTHORPE, F.L. (1962). Drought resi-stance of crop plants.FieTd Crop Abstr. Þ: I7I-179.

LORD, K.A

LOVETT, J

l'lARTIN, G

MAYR, lì.H

McCREE K

}48]DNER,

MEYER, \^/

MICHN]Etr'/ICZ,

MILBORRO\,,/,

MILBURN, J

MISHRA, D

M]SHRA, D

MITCHELL,

113

anrl \r/IIEEI-ER, A.\ü. (f98l). Uptake ancl novement-- of 14c-c,lilornie-

quat chloricle applj ed to leaves of barley ancl rsheat 'J, )ixp. tsot. 32; 599-603.

V. ancl CAIiPIlEl,l,, D.A. (f973). llf f ects of CCC ancl noj.sture sLresson sunflower. Expl . Agric. 9'. 329-336,

C. anct LOPUSIIINSKY, \^/. (1966). Eff ect of N-dimethyl amino-succinamic acid (8-995), a gro\Ì'th retardant, on drought Lolerance'Nature 2O9: 216-217.

. and PAXTON, R.G. (1962)' Quaternar)¡ amrnonium bases in the tomatopl ant . Experientia XVTII ( 10) : 44O-4/+I .

.J. (L914). Changes in stomatal response characteristics of grainsorghum produced by water stress during growth.Crop Sci.. 14: 273-278.

H. and I'{ANSFIELD, T.A. (1968) . 'PhysioTogy of Srontata' .

McGraw-FIj-1I, London.

S. and GREEN, G.C. (198f). Compari-son of stornatal. action of orange,soybean and wheat under field conditions.Aust. J. PTant Physiol. Bz 65-76.

MrYAMOTO, T. (1962)trimethvalues

l'4. and KENT'ZER, T. (1965). The increase of frost resistanceof tomato plants through application of (2-chloroethyl trimethyl-ammonium cirloride) CCC. Experientia XXIz 23O-23I.

B.V. Q979). Antj-transpi-rants and the regulat-j-on of abscícicacid content. Aust. J, PTant PhysioT. 6,2 249-254.

.4. (Igl9). Water flow in plants. Longman, London and Nerv York.pp. 1 -2I.

and PRADHAN, G.C. (1968). Delayed r,rilting of tomato plants by

chemical closure of stomata. Bot. tlag" Tokyo. 81: 219-225.

and PRADHAN, G.C. (L972). Effect of transpiration-reducingchenicals on grotvth flowering and stomatal opening of tomatoplants. PTant Physiol. 50: 27I-214.

J.W., \^IIR\,JILLE, J.W. ancl \'/EIL, L. G949). Plant groivth regulatingproperties of some nicotinium compounrls. Science 110: 252-254.

Effects of the seed treatnent with (2-chloroethyl)amnoni,unl chloricle on the resjstance to high ar]d lor+ pllsoil s j-n wheat seerllings . Naturu'isso 492 371 .

rfof

MTZRAHI, Y., BLUMENFIILD, A. anrl RICII}4ONI), A.E. (l-970). Absc.Lcic acid and

transpiratj.on i,n leaves in relation to osmotic rooL stress.PTant PhysioT. 46t 169--17L"

IL4

I'îONSELISE, S.P., GOREN, R. and HALE\¡Y, A"ti' (196fi) 'Cycocel and Benzotlriazole oxyarceLate on

of lemon Lrees. Proc" Amer" Soc' Llot-t'

J.M. (1917a). changes in diffusive conduc-t'-ance ancl wat-er potentialof tvheat plants before ancl after anthesj-¡s'Aust. J. PTant Physiol. 41 75-'86'

J.M. (1977b). Differences in osrnoregulation betrveen wheat genotypes'Nature 2JOz 234-235,

I,{ORESHET, S. (Lgl5). Effects of- phenyl-mercurj-c acet-ate on storüatal anrl

cuticular resistance tã transpiration. Nerø PhytoT" l5z 47-52'

NAGARAJAII, S. (1978). Sorne differences in the responses of stomata of the

tr,¡o leaf surfaces in cotton. Ann. tsot. 422 II4I-I741 "

NINNEÌ"IANN, H., ZEEVAART, J.A.D., KENDE, FI. and LANG, A. (1964). The plantgrorvth retardant CCC as inhibitor of gibberell in biosynthesis inFusariun Tiforne. Planta þLr 229-235'

PALEG, L.G., KENDE, Il., NINNEMANN, tl. and LAllG, A. (1965). Physiologicaleffectá of gibberellic acid. VIII Growth retardants on barleyendosperrn. PTant PhysioT. 40: 165-169'

PALFI, G. and JUHASZ, J. (197f). The Lheoritical basis and practi'ca1application of a new method of se1ection for deterrnj-ning \^/ater

deficiency in plants. P7. and Soil 342 503-507'

PARKER, J. (1968). Drought resistance-rnechanisms'and Plant GrowtÌ-t'. Ed. Kozlowski, T'T'New York and London. PP. 195-234'

PASSIOURA, J.B. (7912). The effect of root geometry on the-yield of 'wheat

gro"ìng iÁ storerl water, Aust. J. Agric. Res. 23, 745-752.

PEMADASA, M.A. (1919). llovements of abaxial and adaxial stomata'New Phyto7.82: 69-80.

PEI',IADASA, M.A. (1981). Abaxial and adaxial stomata behaviour and responses

to fusicoccin on isolat.ed epidermis of Contnefina connunis L.New PhYtoT. B9z 373-384.

LAI'4BETH, V.N. and IIINCKLEY, T.M. G979).f orn and l-evel on ion concentrat-ions, waterblossom-end rot incidence in t.omato.J. Aner. Soc. Hort. Sc:.. i03: 265-268.

I1f f ects of B-9 ,

florver bud initiati,onSci" 89: 195-200.

I'ÍORGAN,

l"lORGAN,

In: 'Water DeficitsVo1.1. Academic Press,

Effects of nitrogenstress, and

PILL, W.G.,

PISARCZYK, J.M. and SPLITTSTOESSER, B. (1919). Response of tomato to Pre-transplanting applications of chlormequat, Daminozie and lìthephon'Hort. Science 14; 263-264.

PLAUT, Z.,IIALEVY, A.H. and SIÌMUELI, E. (1964). The effect of growt-h-

retar:áing chemical.s on growth and transpiration of bean plantsgro\vn under var j-ous irri-gation regirnes 'Israel, J. Agric" Res" 14: 153-158'

115

PI,AUT, Z arìd I-IALEVY, A.ll" (1966). Regeneration afLer wi.1t-jng, growth and

yì eld of rvheatt plants, as af f ected by trvo grovrt--h-retardingcompounds . PhysioT" Plant 19t 106/+"1012

PRETSON, \,/.H.Jr. and l,,INK, C.B. (1958). Dwarf ecl progeny pr-ocluced byplants treated lvj.th several quaternary ammoniun conlpoullds.PTant PttysioT. 33: (Sirppl . ) xli x.

RAJAGOPAL,

RASCHKB, K

RASMUSSEN,

v. and ANDIÌRSEN, A.S" (1978). Does abscicic acid influenceprolr'_ne accunulation -Ln slressed leaves? PTanta l4?z B5-BB.

Gg15)" Stornatal action , Ann. Rev' Pl'ant Pltltsiç]'' ZQ' 309-340'

o"s. (L976). Abscicic acid level in tonato leaves al-ter a

long period of rvilLing. Physiol, PTant 3þ: 2OB-212.

E. (1962). The inhibj tory effect of 2-chl-oroethyltrirnethyl-arnnìonium chloride treatnent on tobacco nlosaic (Tl"lV) multiPli-cation. Plant Disease Rep. 46-t 170.

and CROZIER, A. (1970). Ccc-induced increases of gibberelljnlevels in pea seecllings. PTanta (Ber7.) 9!; 95-106.

G.A. and GREEN\{AY, H" (1973). Effect-s of CCC on drought resistauceof 'Iriticunt aestivun L. and Zea nays L. Ann. Bot. 31'. 929-934.

RAWLINS, T

REID, D.M

ROBERTSON,

ROBINSSN, J.B.D. (1975) . The inf luence of some grorvttr-rgulating conpoundson the uptake, translocation and concenLration of mineralnutrients i.n plants. Ilort. Abstr. 422 6f 1-618.

RUSSEL, S.L. and KIMMINS, \'1.C. (1912). The effect of (2-chloroethyl)trirnethylammonium chloride on the grovrth of barley'Ann. Bot. 36¡ 785-790,

SABINE, S. ancl DöRFFLING, K. (1981). Evi.dence againsL an intermediary roleof abscicic acid in stonatal closure inducecl by phenyl mercuricacetate and farnesol. Physi-ol- PLan¿ 53: 487-49O'

SACHS, R.M., LANG, 4., BRE'IZ, C.F. and ROACll, J. (f960). Shoot hj-sto-genesis : subapical meristenatic activity in a caulescent plantand the action of giberellic acid and A110-1618.Aner. J. Bot, 47: 260-266.

SACHS, R.M. ancJ KOFRANEK, A.M. (1963). Comparative cytohistological studieson inhibition and pronotion of stem growth in Chrysanthenunnarifoliun. Aner. J. Bc¡t. 50: 172-779.

SALISBURY, F.B. ancl ROSS, C. (1969). 'P¡.ant Pltysi.ology'. WadsworthPublishing Cornpany Inc. , Belnont, California. pp. 52-17 '

SAMPSON, J. (1961). A method of replicating dry or moist surfaces forexamination by light microscopy. Nature I9Iz 932-933.

S?rIrICHEZ-DIAZ, 11.F. and KRAMER, P.J. (1971 ). ]Jelraviour of corn and sorghum

under water st.ress and during recovery. P7ant. PhysioT. 48: 613-616.

SANCIIEZ-DIAZ, l'{.F. and KRAlvlER, P.J. (L973). Turgor differences and waterstress j-n na|ze and solghun lea,ves during drought and recovery.J , lixP. Bot " 2t, z 511-515 .

116

SCIINIìIDER, B.F" (1967). Conversion of plant growth retardanttrimethyi_anmonj-urn c.hlorirle to choline in shoots ofand barley. Can', .J. Biochen. 452 395-400'

sltANTZ, H.L.

SHEPHERD, W.

( 2-ch1or-oeth¡,1)chrysantl-iernuni

Qg27). Droughr- resi-stance and soil noisture" EcoTogyBt 145-157.

(1976). Indices of plant \,/ater status : sone comparisons r¿il-,hin

and betrveen crop species. J. App.l. Eco1. 13: 2'05-209.

ASPINALL, D. and PALEG, L.G. (1973). Stress metabolism. IV'The influence of (2-chlorethyl) trimet-hylammonium chloride and

gibberellic acid on the grorvth and pr:olì-ne accumula.tion of wheatplants during water stress . Aust. J. bio7. .Sci. 262 17-86,

srNGH, T.N.,

STNGII , T. N ., ASPINALL, D. and PALBG, L.G. (L912). Proline accumulation and

varietal adaptability to drought in barley : a potential meta-bolic measure of drought resistance.Nat. New Bio7. 2362 188-190.

SINHA, A.K. and \^IOOD, R".K.S. (1964). Control of l/erticiTTiun wiTt of torìatoplants rvith'r'Cycocel'r (2-.h1o.oethy1 trimethylarnmoniunt chloride).Nature 2O2: 824.

SLATYER, R.O. and BIERHUIZEN, J.F. (T964). The influence of several tra[rspi-ration suppressants on transpiration, photosynthesis and water use

efficiency of cotton leaves. Aust. J. bio1. Scj. 14: 131-146.

SQUIRE, G.R. and JONES, M.B. (1971). Stuclies on the mechanisms of action ofthe antitranspirant phenylrner:curic acetate, and its penetratj,oninto the mesophyll " J. E*p. Bot. 222 980-99I.

STEI¡/ART, C.R. (1980). The mechanism of abscicic acid-i,nduced prolineaccumulation in barley leaves, PTant Physiology 662 230-233,

STgDDART, J.L. (1964). Chemical changes in LoTiun tonulentun L. afterLreatment with (2-chloroethyl) trimethylanunoniurn chloride (CCC)

J. Exp. Bot. 16: 604-613.

some plant(Boyer).

TAHORI,4.S., ZBIDLIIR, G. and HALEVY, A.H. (1965a). Effect of some plantgrowth retardants on the feeding of the cotton leaf worn.J. Sci. Fd. Agric. 16: 570-572.

TAHORI, 4.S,, ZETDLER, G. and LIALBVY, A.H. (1965b). EffecL of sorne plantgrowth retarding compounds on three fungal diseases and one

viral disease. PTant Disease Rep. 492 715-777.

TAL, l'{. and IMBER, D. (f970). Abnormal stonatal behavior¡r and hormonalimbalance in flacca, a wilty mutant of tonaLo. II. Auxin and

abscicic acid-lj.ke activity. PTant Phyúo7. 462 373-376.

TOLBERT, N.E. (1960a). (2-chloroethyl) tr:imethylammonium chloricle and

relatecl conpounds as plant growth substances. I. Chemical' structure ancl bioassay. J. Biol" Chen. 2352 475-479'

TOLBERT, N.E. (1960b). (2-chloroethyl) trimethylarnmoniun chloride and

related compounds as plant gror,rLh substances. II. Effect orl

growth of rvheat . PTant Physiol. 35: 380-385.

TAHORI, 4.S., HALBVY, A.H. and ZETDLER, G. (1965). Effect ofgrowth retardants on the oleander aphid Aphis nerii

' J. Sci. Fd. Agric. 16: 568-569.

1r1

TURNER, N C. (I914). Stornatal behavjour and k'ater status in maize, sorghumand tobacco undet: field conditions. II. AL lor.v soil t+at-erpotential . P-lant Physiol. 53: 360-365"

C., BEGG, J.E" and TONNET, l'1.L. (1978). Osmotic adjustment ofsorghum ancl sunflo\ver crops i.n respouse to rr¡ater cleficits andiLs influence on the water pot-entì,al at which stomata c1ose.Aust. J. PTant Pltysiol. 5: 597-608.

C. (L919). Drought resistance and adaptation to water deficitsin crop p1-anLs. In: tstress PhysictJog)' in C|'op PTarttst .

Eds. Mussell, H. and Staples, R.C. hliley-fnterscience, Neiv York.pp. 343-312.

TURNER, N

TURNER, N

TYREE, M.T. (1976). Negative turgor pressure in p1ant ce11s : fact orfallacy? Can, J. Bot. 54. 2738-2746"

. (1969). The effect of CCC on grotvth and gibberellin contentof tomato plants. Neth. J" agric" Sci-. 772 lB3-183.

H.F. Q964). Effect of (2-chloroethyl) trimethyl'ammoniumchloride on tlte rate of increase of the cabbage aphid(Brevicoryne brassicae L.). Nature 2OJz 946-948.

VAN BRAGT, J

VAN EI"IDBN,

VAADIA, Y. G916). Plant hormones and water stress.Phil,. I'rans. R. Soc. London (Series B). 2132 513-522.

IVEATHERLY, P.E. (1950). Srudies in the water relatj,ons of the cotton plant-I. The fielcl measurement of water deficits in leaves.Nerv Phytol . t¡9 z BI-97 .

1^/EATI-IERLY, P.B. (1970). Some aspects of water relations.Adv" Bot. Res. 3: 17I-206

\^/IR\\¡ILLE, J.\rl. and MITCHELL, J.hl. (1950). Six new plant-gr:owth-inhibitingcompounds, Bot. Gaz. 111: 49I-494.

IltITTi,lER, S.H. and TOLBERT, N.E. (f960). (2-chloroethyf ) trimethylanmoniumchloride and related compounds as plarrt growth substances.III. Effect on grorvth and flowering of the Lomato.Aner. J. Bot. 472 560-565.

WÜUSCUp, U. (f969). Grorqrh retarcling and stimulating effects of CCC on

Antirrltinun najus L. Pfanta (Berl'.) 85: 108-110'

ZEEVAART, J.A.D. (1964). Effects of the grorvth retardant CCC on floralinitiaLion and growth in Pharbitis ni7 -

PTant Physiol, 39; 4O2-4O8.

ZEEVAART, J.A.D. (1965). The grorvth retardant CCC as an inhibitor ofgibberelli.n production in irnmature PharbitisPTant Ph¡,sio1. 40: lxxv.

ZEEVAART. J.A.D. (1966). Reduction of the gibberellin content of. Pharbitisseeds by CCC ancl after-effects in the progeny.PTant Pltysiol , 412 856-862.

ZEI,.ITCI],

ZELITCI{,

118

I . ( 1961) . Biochem_'r-ca1 control, of st onartal opcning in lea v es .

Proc. Nat. Acad. Sci. 4lz )'423-L433

I. and I^/AGGONIÌR, P.B. (f962.). Bffect of cl-reniical. control of stomataon trauspiration and photosyrrthesis.Pt'oc. Nat" Acad. Sci " 4Bz I101-1108.

ZELITCH, I. (f969). Stornatal cont-.rol. Anrt. Rev. P7. Physiol. ZQz 329-350'

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APPENDIX I

FIG.1: Photograph showing Lhe method of deter-

minatj,on of transpiration rate in excised

leaves as described in Section 1I.8.3.

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APPENDIX II

Dete rnination of stomatal density

The number of stomata per unit leaf area was deternined

from leaf surface impr:ints following the method described in

Section II.B.2.

Results from Table 1 reveal that at 3 days after ccc

treatment the number of stomaLa/unit area of leaf in the more

matured 4th leaf and the younger 6th leaf were unaffected by CCC

but inherently there were nore stcirnata on the abaxial surface

than the aclaxial. However, after 10 days, the stomatal density

increases markedly in the CCC-treated plants, especially in the

younger leaves, which could be an effect resulting from the re-

duction in leaf area due to CCC treatment. As discussed in

Section III.3.3, CCC reCards the growth of developing or newly

formed leaves. \^/ith a decrease in leaf area but unchanged total

number of sl-omata, the number of stomata/unit leaf area subse-

quently increases.

An attempt to measure Stomatal apperture was not successful

since the stomata appeared closed as shown in Figures 2 and 3.

The closure of stomata might have been caused by the silicone

rubber used to make tl-re epiderrnal impri-nts, as it was deleterious

to the leaves. Figures 2 and 3 also show the increase in

abaxial stomatal density as compared to adaxial and the lack of

effect of CCC on stomatal denslty of Uhe 4th leaf at 10 days

after CCC application.

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TABLE 1: Number of stomat u/^ 2.

Control F.C.

Control stress

ccc F.c.

CCC stress

L,S.D.57.

CCC

hlater stress

CCC'\water stress

3 days after CCC treatment4th leaf 6th leaf

Adaxial Abaxial Adaxial Abaxial

75 .70 186.00 136.00 319.00

80.30 188.00 13s.00 316.00

10 da4th leaf

Adaxial Abaxial

r CCC treatment6th leaf

Adaxial Abaxial

93.00

1 19.30

98.30

106.30

2t6.OO

257.O0

233.00

250.00

r22.70

r49.30

176.70

r52.70

29.t+9

363.00

398. O0

420.00

398.00FN)ts

s1 .63

FIG.2

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Photographs showing stomatal distri-bution on Lhe adaxlal surface.

Imprints of the 4th leaves were niade

10 days after CCC treatment and the

leaf tissues v¡ere from well-wateredplants.

The stomata appeared close and,

therefore, it was impossible to measure

stomatal aperture.

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' ccc

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FIG.3: Photographs showing stomatal disLributionon Lhe abaxial surface.

ImprÍnÈs of the 4th leaves were made

10 days after CCC treatment and the leaftissues were from well-watered plants.

Stonatal density of the abaxial surfaceappears higþer than the adaxial.However, stomatal aperture could not be

measured as the sLomata appeared close.

I

CONTROL

' ccc