High temperature effects on floral development and vegetative growth of Chrysanthemum X morifolium and the involvement o...

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High temperature effects on floral development and vegetative growth of Chrysanthemum X morifolium and the involvement of plant growth substances
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Whealy, Catherine Anne
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Chrysanthemums -- Effect of high temperatures on   ( lcsh )
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Thesis (Ph. D.)--University of Florida, 1987.
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Includes bibliographical references (leaves 62-75).
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by Catherine Anne Whealy.
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Vita.

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HIGH TEMPERATURE EFFECTS ON FLORAL DEVELOPMENT AND
VEGETATIVE GROWTH OF CHRYSANTHEMUM X MORIFOLIUM AND
THE INVOLVEMENT OF PLANT GROWTH SUBSTANCES




By


CATHERINE ANNE WHEALY


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY




UNIVERSITY OF FLORIDA


1987




























This dissertation is gratefully dedicated

to my parents, Ruby and Roland, and

to my best friend, Bryan.














ACKNOWLEDGEMENTS


My most sincere appreciation is extended to Dr. Terril Nell for his

enduring support, patience, and inspiration. Special thanks are given to Dr. Jim

Barrett for his guidance and astute criticism. Gratitude is extended to my

esteemed committee members, Dr. Jerry Bennett, Dr. Hilton Biggs, and Dr.

Dewayne Ingram for their counsel and encouragement.

I gratefully acknowledge Mr. Carl Scharfenberg of Yoder Brothers, Inc.

for conceiving this research and for his insightful perceptions and direction.

Gratitude is extended to Dr. Jack Downs, Dr. Judy Thomas, Mr. Pete

Ferril, the Phytotron staff, and Dr. Roy Larson for their assistance with our

controlled environment studies.

I am grateful to Ornamental Horticulture staff members: Mrs. Jackie Host,

Mr. T.D. Townsend, Mrs. Nancy Philman, Ms. Carolyn Bartuska, Ms. Deb Gaw, Ms.

Ria Leonard, Mr. Bob Weidman, and Ms. Justine Wetherington for their technical

support, integrity, and cherished friendship.

Evaluation and direction by members of the Ornamental Horticulture

faculty: Dr. Tom Sheehan, Dr. Charlie Guy, Dr. Dennis McConnell, Dr. Frank

Marousky, and Dr. Mike Kane are gratefully acknowledged.

The IFAS Electron Microscopy Facility kindly provided use of equipment.

Appreciation is expressly extended to the American Florist's Endowment

and to Mr. Walter Preston, Manatee Fruit Co., for their financial support.















TABLE OF CONTENTS


Page


ACKNOWLEDGEMENTS .


LISTOFTABLES ................. ...... ....... vi

LIST OF FIGURES . .... .. .. .vii


ABSTRACT .

CHAPTERS


I INTRODUCTION ...........


II REVIEW OF THE LITERATURE


S. 1


. . . 3


III HIGH TEMPERATURE EFFECTS ON FLORAL DEVELOPMENT AND
VEGETATIVE GROWTH OF CHRYSANTHEMUM X MORIFOLIUM. 26

Introduction . . . 26
Methods and Materials ................... 27
Results and Discussion ............... ........30
Effect of High Temperature on Vegetative Growth ... 30
Effect of High Temperature on Floret Initiation and
Differentiation . . 33
Effect of High Temperature on Flowering. . .. 33

IV INVOLVEMENT OF SUPRAOPTIMAL TEMPERATURES AND
PLANT GROWTH SUBSTANCES ON DEVELOPING
INFLORESCENCES OF CHRYSANTHEMUM X MORIFOLIUM .43

Introduction . . . 43
Methods and Materials ..................... .. 45
Exogenously Applied Plant Growth Substances .... 45
Enzyme-Linked Immunosorbent Assay for Zeatin Riboside 46
ACC Determination ................ ... 48
Results . .. . . 49
Exogenously Applied Plant Growth Substances .... 49
Enzyme-Linked Immunosorbent Assay for Zeatin Riboside .55
ACC Determination ................. .. 55
Discussion ...... . . 55

V SUMMARY AND CONCLUSIONS. . . .60








REFERENCES. . .... . .... .62

BIOGRAPHICAL SKETCH ...... ........ .. ... .........76














LIST OF TABLES

Table Page

3-1. Duration and timing of high-temperature treatments that
were used on 'Orange Bowl' and 'Surf' chrysanthemums. ..... 29

3-2. Effect of high- (300/260C) and low- (220/180) temperature
treatments during short days on leaf, stem, and inflorescence
final dry weights of the lateral shoot for'Orange Bowl'
chrysanthemums ................... ...... 31

3-3. Effect of high- (300/260C) and low- (220/180) temperature
treatments during short days on leaf, stem, and inflorescence
final dry weights of the lateral shoot for'Surf' chrysanthemums 32

3-4. Effect of high (300/260C) and low (220/180) temperatures
during short days on selected growth parameters of 'Orange Bowl'
and 'Surf' chrysanthemums . ..... 34

4-1. Effect of low (320/21 C) or high (380/270) temperatures
during short days and exogenously applied chemical treatments at
stage of floret initiation on lateral stem length and inflorescence
diameter on 'Orange Bowl' chrysanthemums. ... ..... 50

4-2. Effect of low (320/21 C) or high (380/270) temperatures
during short days and exogenously applied chemical treatments at
stage of floret initiation on number of short days to stage of
showing-color and open-flower on 'Orange Bowl' chrysanthemums .52

4-3. Effect of low (320/21 C) or high (380/270) temperatures
during short days and exogenously applied chemical treatments at
stage of floret initiation on number of florets and noninvolucral
bracts per inflorescence on 'Orange Bowl' chrysanthemums .53

4-4. Effect of low (320/21 0C) or high (380/270) temperatures
during short days on endogenous zeatin riboside content of
'Orange Bowl' chrysanthemum inflorescences harvested at
developmental stages: 1) floret initiation (3-4 rows of florets
initiated) and 2) floret differentiation (3-6 rows of florets
with differentiated perianth) . ..... 56














LIST OF FIGURES


Figure Page

3-1. Effect of high (300/260C) and low (22/180) temperatures
during short days on number of rows of florets initiated (A)
and number of rows of florets with differentiated perianth (B)
for'Orange Bowl' chrysanthemums . ... .. 35

3-2. Effect of high- (300/260C) and low- (220/180) temperature
treatments during short days on time to stage of showing-color
and open-flower for'Orange Bowl' and 'Surf' chrysanthemums. 36

3-3. Meristem of 'Orange Bowl' chrysanthemum exposed to
high (300/260C) temperatures for first 4 weeks of short days 38

3-4. Secondary inflorescence formed on a receptacle of
'Orange Bowl' chrysanthemum exposed to high temperatures
(300/260C) from start of short days until week 10. . ... 39

3-5. Effect of increasing duration of high (300/260C)
temperatures during short days on mean number of florets per
inflorescence for'Orange Bowl' chrysanthemums. . .40

4-1. Effect of low (320/21 C) or high (380/270) temperatures
during short days on endogenous ACC content of 'Orange Bowl'
chrysanthemum inflorescences harvested at developmental stages:
1) floret initiation (3-4 rows of initiated florets), 2) floret
differentiation (3-6 rows of florets with differentiated perianth),
and 3) floret development (all florets with differentiated
perianth) . .. .. .. .. 57














Abstract of Dissertation Presented to the
Graduate School of the University of Florida in
Partial Fulfillment of the Requirements for the
Degree of Doctor of Philosophy



HIGH TEMPERATURE EFFECTS ON FLORAL DEVELOPMENT AND
VEGETATIVE GROWTH OF CHRYSANTHEMUM X MORIFOLIUM AND
THE INVOLVEMENT OF PLANT GROWTH SUBSTANCES

By

Catherine Anne Whealy

August, 1987


Chairman: Dr. Terril A. Nell
Major Department: Horticultural Science


Pinched plants of Chrysanthemum X morifolium Ramat. 'Orange Bowl' and

'Surf' grown in a chamber maintained at 220 day/180C night were transferred to

300 day/260 night at the beginning of week 1, 3, 5, or 7 after start of

photoinduction period (15-hour nyctoperiod). Plants remained at high

temperatures for 2, 4, 6, 8, or 10 weeks and then were returned to the 220/180

chamber. Exposure to high temperatures during the first 4 weeks of short days

caused an increase in number of nodes, leaf area, stem length, and dry weight of

leaves and stems. Rate of floret initiation and perianth differentiation decreased

when plants of 'Orange Bowl' were exposed to high temperatures during the first

4 weeks of short days. 'Orange Bowl' exposed to high temperatures for 10 weeks

from start of short days flowered 12 days later than plants grown at lower

temperatures and formed bracteate buds. Flowering of 'Orange Bowl' grown at








220/180 during the first 4 weeks of short days, then transferred to high

temperatures, was not substantially delayed and inflorescences developed normally.

Flowering was delayed 3 days when 'Surf' was exposed to high temperatures for 8

weeks from start of short days. Exposure to high temperatures did not cause

bracteate bud formation in 'Surf'. With both cultivars, increasing the duration of

high-temperature exposure increased time to flowering. Exogenous applications

of plant growth substances during floret initiation accentuated or diminished the

teratological modifications of supraoptimal temperatures. Cytokinins, gibberellins,

and inhibitors of ethylene biosynthesis promoted noninvolucral bract formation on

initiated receptacles of 'Orange Bowl' plants. Ethephon (2-chloroethylphosphonic

acid) treatments reduced number of noninvolucral bracts. Endogenous zeatin

riboside content of induced inflorescences increased with advancing stage of

development and increased ambient temperature. Level of 1-aminocyclopropane

carboxylic acid (ACC) increased during stage of early floret diffentiation in plants

exposed to optimal production temperatures, but supraoptimal temperatures

suppressed ACC increase. It appears that gibberellin and/or cytokinin level,

activity, or sensitivity may increase in response to supraoptimal temperatures in

chrysanthemum causing abnormal inflorescence development, and ethylene appears

to be necessary for normal inflorescence development.














CHAPTER I
INTRODUCTION

The chrysanthemum is a herbaceous perennial that has been in cultivation

for over 2500 years (30). Today, cultivars of Chrysanthemum X morifolium Ramat.

(12) are some of the world's most popular cut flowers and potted ornamental

plants. This popularity is due in part to the ability to control year-round

commercial availability of the flowers by manipulating daylength and by providing

an optimal environment for growth and floral development.

Temperature, irradiance, and photoperiod are the major factors affecting

the rate of initiation and differentiation of chrysanthemum inflorescences.

Improper photoperiods, low irradiances, and temperatures above or below an

optimum can inhibit or delay flowering and may result in abnormal inflorescence

formation. It has long been recognized that the effects of temperature on

chrysanthemum flowering can be as dramatic as those associated with photoperiod

(145). During the winter, irradiance is usually the limiting factor determining

rate of floral development; in the summer, high temperature may be limiting and

flowering can be delayed. Rate of floral development can be optimized only by

proper photoperiod, high irradiances, and controlled temperatures.

In the midwestern and southern United States, high summer temperatures

are common, often resulting in delayed flowering and/or poor quality

chrysanthemums. This phenomenon is commonly referred to as "heat delay."

Although a significant amount of research has been conducted on the influences

of temperature on chrysanthemum flowering, detailed information concerning

specific developmental stages and the physiological responses affected by








2

supraoptimal temperatures is not available. The objectives of these studies were

to determine the developmental stages most sensitive to high temperatures, to

determine the effects of high temperature on vegetative growth, and to

investigate the hormonal changes associated with high temperature exposure.














CHAPTER II
REVIEW OF THE LITERATURE

Chrysanthemum X morifolium Ramat. is a member of the family

Compositae (Asteraceae), tribe Chrysantheminae (12). The inflorescence is a

raceme consisting of a capitulum of marginal ligulate or ray florets pistillatee) and

center tubular or disc florets (hermaphrodite) which are initiated centripetally and

arranged spirally on the capitulum (110). An involucre of bracts subtends the

capitulum which is ebracteate (11). Rate and modifications of capitulum and

floret development are affected by environmental conditions and/or chemical

treatments (30,41).

Chrysanthemum is a short-day (long-night) plant (6,72) which naturally

flowers in the autumn or winter (30,131). Allard (6) and Garner and Allard's (72)

discovery that flowering could be manipulated by photoperiod was utilized by

Laurie (109) to extend the natural flowering season of the chrysanthemum.

Subsequently, Post (138) demonstrated that chrysanthemums could be flowered

year-round provided that the daylength was less than the critical photoperiod, the

night temperature was controlled, and the cultivars selected carefully.

Cultivars are classified for natural or year-round flowering by response

groups which represent the number of weeks of photoinductive short days to

attain flower maturation (35). This classification is arbitrary as temperature and

irradiance levels affect rate of flower development (16,26). Floral development in

chrysanthemum can be divided into three phases: receptacle initiation, floret

initiation, and floret development (differentiation). The difference in response








4
times between cultivars is a function of the rate of floret development, not of

receptacle or floret initiation (62).

As a determinate plant, the number of leaves formed prior to the

inflorescence is indicative of the rate of inflorescence initiation (38). The onset

of flowering (receptacle formation with initiation of involucral bracts [31]) can be

observed after three to six consecutive short days depending on cultivar

(34,85,142). This response to photoperiod is quantitative (41) since receptacle

initiation will eventually occur under noninductive photoperiods (39,40) after the

attainment of a critical apical meristem size (36,85). This autonomous induction

can be quantified by the initiation of a genetically determined number of leaves

(long-day leaf number) characteristic of cultivars (39,45), depending on

temperature and irradiance levels (40). Subsequent inflorescence development is

quantitative in early-flowering cultivars, summer or garden cultivars (shorter

response time) (107,160), but qualitative for late-flowering cultivars, autumn and

winter cultivars (longer response time) as anthesis is not attained in these

cultivars exposed to noninductive photoperiods (107,142,158).

The critical photoperiod for floret initiation is longer than for floret

development (28,70,139), and is dependent on cultivar (response group) (70),

irradiance (150), and temperature (28). Generally, the critical photoperiod for

floret initiation and development increases with decreasing response times

(28,70,178). Increasing the minimum night temperature from 100 to 26.50C will

increase the critical photoperiod for initiation but decrease the critical

photoperiod for floret development (28).

Apical inflorescences of chrysanthemums produced under inductive

conditions are referred to as "terminal buds"; these inflorescences have fully

differentiated florets and are subtended by reproductive lateral shoots (34,137).








5
Exposure to noninductive photoperiods results in arrested and/or distorted

development of the apical inflorescence, a loss of apical dominance, and

resumption of vegetative lateral bud growth (30,135,157,158). The resulting

arrested and malformed meristems are commonly referred to as "crown buds"

(34,135). Crown buds are modifications of terminal buds in which receptacles are

initiated, but floret initiation and subsequent floret development are inhibited

(34,134,158). Thus, crown buds will form if the photoperiod is extended beyond

the critical photoperiod after inflorescence initiation (137,141); on late-flowering

cultivars exposed only to long photoperiods; on plants exposed to long

photoperiods during which light intensity is limiting (134); or when photoperiods

are short enough for receptacle initiation, but too long for floret development

(139). If plants are returned to inductive photoperiods, the inflorescences will

continue to develop (143,158). Manipulating crown bud formation can be used to

improve spray formation (103,140) and to eliminate the need for pinching of cut

chrysanthemums (141,142). However, intercalating 10 long days after 11 short

days to increase peduncle length on cut chrysanthemums caused bract formation

on the receptacles of axillary meristems of 'Hurricane' and 'Statesman', additional

disc florets on 'Flame Belair', and secondary inflorescences on 'Pinocchio' (104).

Teratological effects have been observed on vernalized plants of 'Indian Summer'

exposed for extended periods to noninductive photoperiods, i.e., bracts on the

receptacle, secondary inflorescences and petaloid stamens (158).

Temperature can adversely affect chrysanthemum flowering and is of

prime importance in commercial scheduling and production of flowers.

Temperatures above or below an optimal temperature increase the number of days

to flower and, thus, the response to temperature is parabolic (56,98). In the

greenhouse, researchers have determined the optimal night temperature to be








6
approximately 160 for a number of cultivars (27,35,144). Flowering of the cultivar

Nob Hill has been shown to occur earlier in plants exposed to 220/180 (day/night)

than in plants at higher or lower temperatures in controlled temperature chambers

(19).

The response of chrysanthemum flowering to temperature is cultivar-

dependent (26,35,56,144,155,176,181). Cathey (26) categorized chrysanthemum

cultivars by flowering response to night temperature. Cultivars were classified as

1) thermonegative-inhibited by high temperatures (maximum 16), 2) thermozero-

unaffected by high or low temperatures (100 to 270), and 3) thermopositive-

sensitive to low temperatures (minimum 160). In general, late-flowering cultivars

are more negatively affected by sub- or supraoptimal temperatures than earlier-

flowering cultivars (144).

Discrepancy exists in the literature on the actual inflorescence

developmental stages affected by temperature. It has been argued that the

response to temperature, similar to photoperiod, is quantitative for floret

initiation and qualitative for development (71,144); or the response is quantitative,

i.e., sub- or supraoptimal temperatures during induction will delay the rate of

initiation and development (56). Post and Lacey (144) hypothesized that high

temperatures "counteracted" the short-day photoperiodic influence on flowering in

the chrysanthemum cultivars Indianapolis Bronze, Indianapolis Pink, Marie de

Petris, and Queen's Lace. They noted that high night temperatures, and to a

lesser extent high day temperatures, had a greater effect on rate of inflorescence

development than initiation of receptacle and florets (144). Thus, cultivars

differed in temperature requirements during inflorescence development, but not

during initiation (144). Cathey (27) observed that initiation occurred with

exposure to night temperatures of 40 to 270; however in the thermopositive








7
cultivar Encore, initiation was accelerated at the higher night temperatures, but

continuous high temperatures delayed subsequent inflorescence development.

However, Cathey and Borthwick (31) in 1958 suggested that initiation was delayed

by increased temperatures. According to these researchers, initiation referred to

the time from the start of short days to visible bud, and development was defined

as the phase from visible bud to open flower (27,144). These designations are

arbitrary and are not indicative of the actual morphological stages. Under

continuous lighting, inflorescence initiation in 'Polaris' was delayed at 280 and 100

compared to plants grown at 160 or 220, and 280 increased the number of leaves

formed below the inflorescence (40). However, continuous lighting is inhibitory to

floret initiation. Under inductive conditions, Hughes and Cockshull (86) observed

that high day temperatures (29.30) increased the rate of leaf initiation on 'Bright

Golden Anne', and receptacle initiation was not delayed. Subsequent inflorescence

development was substantially delayed with a decrease in inflorescence dry weight

in plants exposed to 29.30 compared to 18.30 day temperature.

In addition to delayed initiation and/or development, high temperatures

enhanced vegetative growth (10,19,40,57,86), increased floret number (10,27),

induced foliaceous bract formation on the receptacle (144), and arrested

inflorescence development precluding anthesis (144). Apparently, the temperature

optimum for vegetative growth is higher than the optimum for reproductive

development (86). Increased ambient temperatures and irradiances accelerated rate

of leaf primordia formation in 'Polaris' (40); and high day and night temperatures

increased number of leaves and stem length during long days on eight cultivars

(57). 'Escort' chrysanthemums had longer internodes and smaller inflorescence

diameters with exposure to increasing temperatures from 150 to 240 during short

days (10). Stem length and leaf area of 'Nob Hill' chrysanthemum increased with








8

increased temperatures (180/140 to 300/260, day/night) in controlled temperature

chambers (19). The heat-induced increase in leaf area is apparently the result of

a higher percentage of dry matter partitioned to the leaves with a concomitant

decrease in partitioned dry matter to the stems (4).

Temperature can have a profound effect on chrysanthemum inflorescence

form and color. 'Geisha', a pink-spider type at 140, developed as a white daisy at

230 (56), and 'Indianapolis Bronze' chrysanthemums grown at 270 night

temperatures faded to yellow (136). Increasing temperatures reduced length and

width of the ligulate florets of 'Escort' and florets were more tubular (10).

Floret number increased with higher temperatures in 'Encore' and 'Escort' (10,27).

Moreover, plants produced under high temperatures developed bracts on the

receptacle in lieu of florets (144). This modification of a terminal inflorescence

is referred to as a bracteate bud, and is distinguished by the presence of

noninvolucral bracts on the receptacle; the absence of noninvolucral bracts is a

distinguishing characteristic of the tribe Chrysantheminae (158). This

characteristic is modified by temperature (144,180) and photoperiod (104,158), and

is evidence of interrupted or otherwise perturbed floral development (16).

The relative contribution of day and night temperatures have been

debated in the literature. Post and Lacey (144) investigated the effect of high

day versus high night temperatures with the following treatments: 160/160,

160/320, 320/160, and 320/320 day/night, and observed that high night temperature

was more detrimental than high day temperatures on flower development, but

concluded that both day and night temperatures are important. Cathey (27) also

concluded that night temperature had a more profound effect on flowering than

day temperature, and thus, averaging of day and night temperatures was not

correlated with rate of flower development on 'Encore'. However, the unequal








9

contribution of night versus day hours was not taken into account in these

studies (27); hence, Cockshull et al. (42) proposed that flowering is equally

responsive to night and day temperatures. Their work suggests that the mean

temperature over a 24-hour period determines rate of flower development (42).

Recently, Karlsson and Heins (98) have used response surface analysis to evaluate

the relationship between day/night temperatures and photosynthetic photon flux

(PPF) on time to flower and plant quality. In their studies on 'Bright Golden

Anne', the average temperature hypothesis of Cockshull et al. (42) was not

supported; i.e., time to flower for reciprocal combinations of day/night

temperatures were dissimilar (98).

High temperatures have been reported to affect flowering in many genera.

Total leaf number increased in seedlings of Nicotiana tabacum L. 'Coker 319' when

exposed to increasing temperatures (180/140 to 300/260, day/night) with a

concomitant delay in flower initiation (170). The photoperiodic response is

profoundly altered in common cocklebur (Xanthium strumarium L.) by night

temperature, as temperatures of 380 suppressed floral initiation under inductive

photoperiods (78). High night temperatures (210) inhibited bud differentiation in

Gardenia veitchii Hort. compared to low temperatures (130 to 160) (101). High

temperature (280/230, day/night) exposure during flower development reduced the

number of petals, stamens, carpels and locules in tomato (Lycopersicon esculentum

Mill.) compared to low (180/150) or intermediate (230/180) temperature treatments

(156). Pretransplant high temperature treatments (250, 300, or 350 for 10 to 20

days) inhibited bolting, i.e. inflorescence stalk elongation, and promoted vegetative

growth in spring-harvested celery, Apium graveolens L. 'Florida 683', grown under

inductive conditions of low temperatures (147).








10

In chrysanthemum, responses to environmental cues have been shown to

be mediated by endogenous plant growth substances. Tompsett and Schwabe (173)

suggested that flowering responses to environmental cues are regulated by

absolute concentrations and interactions of endogenous plant growth substances.

However, variations in levels of endogenous plant growth substances hypothesized

to be the promoters or inhibitors of flowering may rather be the consequences of

floral induction.

Bioassays have indicated that 'Shasta' chrysanthemums contain more

growth promoting substances (auxins) when grown under long days, and contain

higher growth inhibitor levels during short days (79). Tompsett and Schwabe

(173) observed that inflorescences of 'Sunbeam' chrysanthemums subjected to long

photoperiods contained higher auxin and abscisic acid levels and lower auxin

oxidase levels than plants subjected to photoinductive short photoperiods.

Gibberellin level increased with exposure to short days (173). Exogenous

applications of IAA (indol-3yl-acetic acid) or NAA (1-napthaleneacetic acid) during

the first 4 weeks of photoinduction delayed flowering while GA3 (gibberellic acid)

applications promoted flowering in chrysanthemum cultivars Yellow Spider, Yellow

Giant Indianapolis, and White Bonnie Jean (165). Daily IAA sprays during short

days inhibited flower bud initiation, as indicated by a 2-fold increase in leaf

number, and inhibited subsequent flower bud development in 'Iceberg' (114).

However, the response to applied auxin is rate-dependent as low or high

concentrations may promote or inhibit flower induction, respectively (37,111).

Induced inflorescences of 'Indian Summer' can be inhibited in their development

by exposing them to long days, low irradiance levels, or by application of auxin

paste (158). Sensitivity to auxin applications decreased with advanced stages of

floret development in 'Hurricane' and 'Indian Summer' (75,158) with ovule








11
formation being the latest stage affected (158). Thus, high endogenous auxin

levels may be responsible for inhibition of floret development of chrysanthemums

exposed to noninductive photoperiods (158).

Exogenous applications of IAA have also been shown to inhibit flower

induction and development in other plant species (76). Auxin activity in common

cocklebur was higher when plants were exposed to noninductive photoperiods and

activity declined with the appearance of floral primordia (47). IAA applied to the

plumules of pigweed (Chenopodium rubrum L.) before or during photoinduction

inhibited DNA synthesis and meristematic activity (161). The axillary meristems

were affected the most, since apical dominance was promoted, and consequently

floral differentiation was inhibited.

In whole root extracts of chrysanthemum 'Polaris', zeatin- and zeatin

riboside-like compounds are the major endogenous cytokinins (100) and were

detectable throughout the flowering process (83). There is no conclusive

evidence, via agar-diffusive studies, that cytokinins are synthesized or produced in

chrysanthemum shoot tissue (124). The principal site of cytokinin synthesis is

considered to be the root tip (15). There is a close correlation between

developmental processes in the shoot tip and the quantities of cytokinins

translocated from the roots (15,53). For example, during flower initiation and

development in Perilla frutescens (L.) Britt., a short-day plant, acropetal

translocation and activity of cytokinins in xylem sap increased to 5 times that of

noninduced plants (15). The synthetic cytokinin, 6-benzylamino-9-

(tetrahydropyran-2-yl)-9H-purine (PBA), applied to chrysanthemum 'Bright Golden

Anne' inflorescences prior to floret initiation increased diameter and fresh weight

of the inflorescence with efficacy decreasing with advanced development (93).

Carnation (Dianthus caryophyllus L. 'White Sim') flowers treated with PBA








12

developed a greater number of petals and secondary growth centers which resulted

in "bullhead" flowers (93). There was not an increase in chrysanthemum 'Bright

Golden Anne' floret number as a result of PBA applications (93). Inflorescence

dry weight and floret number increased with BA (6-benzyladenine) treatments to

inflorescences of Leucospermum sp. R. Br. 'Red Sunset' applied during induction

(129). Jeffcoat (93) hypothesized that PBA may be enhancing transport of

assimilates to the inflorescence or affecting the water balance in the

inflorescence. Indeed, cytokinins may be responsible for changes in the import

ability of strong sinks such as inflorescences. Assimilate accumulation in the

shoot apices axes increased and inflorescence development was promoted in

response to short-day photoinduction, removal of youngest leaves, or PBA (N-

benzyl-a-[tetrahydro-2H-pyran-2yll-adenine) treatments in Bougainvillea sp. Comm.

ex Juss. 'San Diego Red' plants (175). Apparently the youngest leaves were

competing sinks for assimilates, and cytokinin applications to the shoot apices or

short days increased sink strength prior to morphological changes (175). Thus,

short days may affect assimilate translocation by affecting cytokinin synthesis

and/or distribution (175,177).

Cytokinins may be inhibitory to flowering. Davey and van Staden (54)

observed a decrease in cytokinin activity of white lupin root exudate, Lupinus

albus L., with the onset of flower development and activity continued to decline

during flower maturation. In tomato, zeatin and zeatin riboside activity decreased

in the root exudate as flower development occurred (53). A dramatic decrease in

the level of cytokinins was observed in buds of common cocklebur in response to

short-day treatment (177), which lead to the hypothesis that the reduction was

requisite to subsequent flowering (82). During early flower development in

Cosmos sulphureus Cav., cytokinin activity in the flower buds was lower than at








13

flower opening (152). A decrease in cytokinin activity in reproductive meristems

or undetectable levels may suggest that cytokinins are being rapidly metabolized.

However, kinetin applications to plumules of pigweed exposed to photoinductive

short days promoted apical meristematic activity stimulating vegetative leaf growth

and suppressing flower differentiation which indicated that the promotion of leaf

growth by kinetin applications correlatively inhibited flower bud development

(163).

There is no simple correlation between flowering and endogenous

gibberellins (133). GA3 applications promoted flower bud initiation in the

qualitative short-day plant Impatiens balsamina L. under noninductive

photoperiods, and repeat applications stimulated subsequent development (128).

However, GA3 applied to apical buds of the long-day plant Fuchsia X hybrida

Hort. ex Vilm. 'Lord Byron' inhibited flower initiation (151). Gibberellic acid

suppressed flower initiation in the short-day plant Fragaria sp. L. Talisman'

maintained under short days, and plants resembled plants produced under long

days (171). In bougainvillea, a short-day plant, gibberellic acid inhibited

inflorescence development similar to effects of noninductive photoperiods, low

light level, and high night temperatures (148). Flower development was delayed in

plants of Euphorbia pulcherrima Willd. (poinsettia) by spray applications of

gibberellic acid under inductive short days (77). Moreover, there may be

inhibitory and promotive effects of applied GA's within a species; GA3 applications

to Pharbitis sp. L. is promotive prior to photoinduction, but inhibitive after

photoinduction (133). These differences may be attributable to tissue specificity

and changes in sensitivity to endogenous phytohormones.

In chrysanthemum, the effect of exogenously applied or endogenous

gibberellins on flowering may involve the distribution of assimilate (121). Jeffcoat








14
and Cockshull (94) found that activity of gibberellin-like substances in 'Bright

Golden Anne' directly correlated with the relative growth rates of the

inflorescence. When inflorescences were at their maximum relative growth rate

(time of highest sink demand), the amount of gibberellin-like substances, as

determined by bioassays, were at maximum levels (94). Gibberellin levels declined

with the decline of the inflorescence's relative growth rate, concomitant with

decline in sink strength. Cathey and Stuart (32,168) showed that gibberellins did

not affect receptacle or floret initiation in short-day treated chrysanthemums as

leaf number prior to inflorescence initiation was unaffected. Applications during

differentiation and later development reduced time to anthesis in 'Shasta' and

'Indianapolis Yellow' chrysanthemums (32,168), and rate of flower bud development

in chrysanthemums grown under inductive photoperiods with initiated florets was

promoted by applications of gibberellins (32,121-123,125,168). Weekly exogenous

applications of gibberellins alone or in combination with BA promoted

inflorescence development in 'Pink Champagne' chrysanthemums grown under

noninductive photoperiods (132). GA and BA effects were synergistic, however,

anthesis was not attained for any treatments, and IAA was inhibitory (132).

Thus, gibberellins are viewed as required promoters of inflorescence development

in chrysanthemums (121).

The growth retardants chlorphonium chloride, daminozide and piproctanyl

bromide delayed inflorescence development but not inflorescence initiation in

chrysanthemum 'Bright Golden Anne' grown under inductive photoperiods (121).

Ancymidol has been demonstrated to inhibit flower development in a dose-response

manner in 'Nob Hill' plants maintained in controlled environments (19). The

effect of growth retardants on reducing gibberellin levels may be a reduction in

translocation of assimilate to the rapidly developing inflorescence, thus delaying








15

development (121). This delay can be reversed by gibberellin applications which

attract assimilates to the inflorescence, and thus accelerate development

(121,123,125).

This apparent role for gibberellins in flower development has also been

demonstrated in rose and carnation. In 'White Sim' carnations, cytokinins (166)

and gibberellins (95) diverted assimilates to the application site, and hence

promoted flower development (81). Removal of the carnation flower resulted in

altered assimilate transport towards the roots. However, applications of GA3 or

IAA to the decapitated stem, redirected the assimilates upward (95). GA3 applied

to receptacles of developing 'Baccara' rose (Rosa sp. L.) flowers increased flower

size, dry weight, and pigmentation (190) probably by augmenting sink strength

(190,193). Endogenous gibberellin levels were lower in leaves of abortive shoots

than flowering shoots (191), and low light and temperature conditions promoting

bud atrophy decreased endogenous gibberellin level (193). Gibberellin applications

to developing rose flowers reduced, and ethephon applications increased flower

bud atrophy (192). Cytokinin (BA or PBA) treatments decreased endogenous

gibberellin activity and resulted in increased incidence of bud atrophy; however,

GA3 applications counteracted the cytokinin effects (194).

Differential reponses to exogenous abscisic acid (ABA) on flowering have

been reported (67). Endogenous ABA levels were higher in chrysanthemum '#3

Indianapolis White' exposed to short days compared to long days (164). Tompsett

and Schwabe (173) postulated that this increased level of ABA may inhibit the

growth of other plant parts during inflorescence development. Daily applications

of ABA did not affect flowering, stem length, or number of nodes on

chrysanthemum 'Fred Shoesmith' (29). However, applications of ABA to induced

pigweed plants were inhibitory to floral differentiation (162).








16

Ethylene or ethylene-releasing compounds promoted flower induction in

pineapple (Ananas sativa L. [48] and Ananas comosus L. Merr. [37]), various

ornamental bromeliads (Aechmea fasciata [Lindl.] Bak., Neoreglia sp. L. B. Sm., and

Vriesea splendens [Brongn.] 'Lemaire') (33), and Belgian endive (Cichorium intybus

L. 'Foliosus') (58); induced flowering in the qualitiative short-day plant Plumbago

indica L. 'Angkor' grown under noninductive photoperiods (130); and enhanced

flower initiation in young apple (Malus sylvestris Mill. 'Delicious') trees (182).

Aechmea victoriana L. B. Sm. plants were induced into flower by ethylene or ACC

(1-aminocyclopropane-l-carboxylic acid) treatments, but plants must be at a stage

of "flower maturity" which coincides with an increase in the tissue's capacity to

convert ACC into ethylene (55). Moreover, flower induction in the bromeliad

Guzmania lingulata (L.) Mez. 'Minor' is positively correlated with endogenous

ethylene production (59). Flower induction was retarded in Bromeliaceae by the

ethylene biosynthesis inhibitor, AVG, L-2-amino-4-(2-amino-ethoxy)-trans-3-

butenoic acid hydrochloride (aminoethoxyvinylglycine) (120). MVG, L-2-amino-4-

methoxy-trans-3-butenoic acid, and AVG delayed flower development in Nemesia

strumosa Benth. (116). In the long-day plant, Spinacia oleracea L., leaf and

whole-plant ethylene production rates were significantly increased by

photoinductive treatments compared to noninductive daylengths (50). ACC was

more concentrated in leaves from noninduced plants, indicating that induced plants

had a greater capacity for ACC conversion (50).

However, in many plants, ethylene inhibits flower formation (189).

Treating peach trees, Prunus persica (L.) Batsch. 'Redglobe', with ethylene-

generating compounds, ethephon and CGA-15281 (Ciba-Geigy, Ltd.), in the late

summer or fall delayed flowering the following spring (49). Ethylene applications

to cotyledons of the short-day plant Pharbitis nil L. Violet' during the inductive








17

dark period were effective in inhibiting floral induction (169). Ethephon

treatments to burley tobacco (Nicotiana tabacum L.) while in seed beds suppressed

premature flower induction and resulted in higher yields (99). Under inductive

short days, foliar applied auxins inhibited flower initiation and development in

common cocklebur (21); while application of triiodobenozoic acid (TIBA) or 2,4-

dichloranisole (DCA) under non-inductive long days induced flower bud initiation

(20). Ethylene inhibits floral induction in common cocklebur (2), thus the effect

of IAA on flowering in cocklebur is probably via its effect on ethylene

production.

Inflorescence initiation and development in chrysanthemum '#3

Indianapolis White' exposed to short days is inhibited by continuous ambient

applications of 1 to 4 ppm ethylene, and intermittent applications resulted in

crown bud formation (172). Plants subjected to ethylene under long or short

photoperiods had thickened stems, shortened internodes, epinastic leaves, and a

loss of apical dominance (172). Ethylene-treated plants which failed to flower

under short days had higher levels of inhibitors auxinn) as determined by

bioassays (172). Low concentrations of ambient ethylene (1 ppm) appeared to

inhibit chrysanthemum inflorescence development more than initiation (146).

Ethephon sprays during short days inhibited inflorescence development and

lowered ABA levels compared to untreated plants ('#3 Indianapolis White') exposed

to short or long days (164). Plants of 'Polaris' treated with 100 or 1000 ppm

ethephon at start of short days were delayed in flower initiation and more leaves

were formed below the inflorescence; and ethephon treatments during long days

inhibited inflorescence initiation to a greater extent than in untreated plants (43).

Rate of leaf initiation and leaf number were increased by ethephon applications;

moreover, apical dominance was modified as leafy axillary shoot development was








18
promoted (43). The number of leaves formed on plants treated with ethephon in

long or short days were similar, indicating that the effect was probably

independent of long-day inhibition of initiation (43). The practical application of

this ethylene effect is use of ethephon to inhibit premature and irregular budding

on garden-type cultivars which bud readily in long days (44).

Ethylene production in vegetative tissues is significantly affected by

exogenously applied plant growth substances (1). It has long been recognized that

auxin promotes ethylene production (197). Tissues treated with IAA contained 100

times ACC and ethylene production was stimulated 500 times compared to

untreated mungbean (Vigna radiata [L.1 R. Wilcz.) hypocotyls (187). In subapical

hypocotyl segments from 3-day old etiolated mungbean seedlings, IAA treatment

increased ACC synthase activity, and BA further enhanced IAA-induced activity

(184). Auxin induces ethylene biosynthesis in mungbean hypocotyls by inducing

ACC synthase and the induction is time- (187) and concentration-dependent in pea

(Pisum sativum L. 'Alaska') seedlings (97) and in mungbean hypocotyls (153). The

conversion of SAM (s-adenosylmethionine) to ACC is the rate-limiting reaction in

ethylene synthesis (183). IAA apparently stimulates de novo synthesis of ACC

synthase since IAA-induced ethylene production is suppressed by inhibitors of RNA

and protein synthesis and there is a considerable lag period prior to IAA-

stimulated ethylene production (1,3,23,153,185). Furthermore, AVG, which inhibits

ACC synthase (5,18), eliminated ACC accumulation and auxin-induced ethylene

production (187). Ethylene treatment inhibited the incorporation of 3H-thymidine

into DNA in pea roots and apices and is thus a possible mechanism by which

ethylene inhibits cell division (7). The inhibitory action of auxin on cell division

in primary meristematic tissues may be attributed to auxin-induced ethylene

production (7).








19

Kinetin applications are synergistic to auxin-induced ethylene production

(24,69). Application of cytokinin (BA or kinetin) was synergistic to IAA-induced

ethylene production in 2- to 5-day old 'Alaska' pea, radish (Raphanus sativus L.

'Early Scarlet Globe'), bean (Phaseolus vulgaris L. 'endergreen Improved'),

cucumber (Cucumis sativus L. 'Boston Pickling'), and maize (Zea mays L. 'Golden

Bantum') seedlings (69). Gibberellin applications did not affect ethylene

production (69). In mungbean hypocotyl segments, cytokinin (BA or kinetin)

applications increased ethylene production with and without IAA (89,105). Kinetin

may be affecting ethylene production by regulating the free endogenous IAA level

via suppression of auxin conjugation in urd (Phaseolus mungo L.) hypocotyl

segments (108), as a higher level of free IAA is correlated with higher endogenous

ethylene production (97,108). However, tracer experiments indicated that the

increase in free endogenous IAA as a result of cytokinin treatments was

insufficient to account totally for the observed increase in ethylene production

(89). It has been proposed that cytokinins may affect ethylene production

directly, similar to IAA regulation (183).

There is evidence of mutually antagonistic effects involving ethylene and

other plant growth substances. In lateral buds released from apical dominance,

and in roots and shoots of seedlings of 'Alaska' pea, cell division was suppressed

at a stage prior to prophase as a consequence of exposure to ethylene; this

inhibition of mitosis was partially reversed by BA applications (7). Coleus (Coleus

blumei Benth.) roots treated with ethephon had decreased cytokinin activity levels

in the root exudate and root growth was inhibited (13). BA treatment of cut

carnation delays senescence by affecting ethylene synthesis and action (46).

Researchers have demonstrated antagonistic responses by gibberellin and ethylene

applications in 'Alaska' pea seedlings (69) and lettuce (Lactuca sativa L.)








20
hypocotyl elongation, invertase activity in sugar beet (Beta vulgaris L.) tissue, and

a-amylase activity in barley (Hordeum vulare L.) endosperm (159). Daminozide

treatments to 'Ingrid Marie' apple shoot tips decreased shoot growth and

increased rate of ethylene production (96). ABA inhibited methionine to ethylene

conversion in 3-day old 'Alaska' pea seedlings (74) and in etiolated mungbean

hypocotyls (105). Furthermore, ABA suppressed IAA-induced ACC synthase

activity and inhibited ethylene production in etiolated mungbean hypocotyls (184).

Therefore, it is probable that auxin, BA and ABA influence ethylene production by

regulating endogenous ACC synthase activity (184).

In response to environmental stimuli, plant growth substances may

regulate ontogenetic development both spatially and temporally via assimilate

transport and distribution (174,179). Sachs and Hackett (148,149) have proposed

that growth substances may control floral initiation and development by regulating

the partitioning of assimilates. Thus, plant growth substances may be acting

indirectly upon flowering by influencing assimilate partitioning by affecting

relative sink strengths, hence, competition between critical meristematic regions

(apical versus subapical) or within the apex rather than directly as morphogenetic

or gene modulators (149). The control may be at the level of regulation of

activities in the sink tissue or at the translocation mechanism (127). Recent

evidence suggests that plant growth substances may directly control phloem

loading of sucrose (52). But, Bodson and Bernier (17) have debated that changes

in carbohydrate status are requisite for flower initiation but are not the sole

controlling factors in flower initiation.

There is ample evidence that the effect of temperature on flowering may

involve photosynthate production, translocation, and/or accumulation. High bud

temperatures (210 to 300) in 'White Sim' carnations increased accumulation of








21
14C-labelled assimilates in the flower at the expense of the stem, and localized

cooling of the bud (100 to 00) decreased the translocation of assimilates into the

flower (80). Rate of shoot apex enlargement was decreased and rates of leaf

formation and growth increased in 'Potentate' tomato seedlings maintained at

constant temperatures of 250 compared to constant 150 (87). The delay in apex

enlargement and the increase in leaf number prior to flowering in response to

elevated temperatures was hypothesized to be a function of competition for

available assimilate, i.e. the sink strength of the leaf primordia being greater than

that of the apex when exposed to higher temperatures (87). Removal of the first

two expanding leaves of tomato seedlings exposed to the high temperature (250

constant) treatment resulted in a rapid expansion of the shoot apex and earlier

flower initiation (88). However, defoliation of low temperature (150 constant)

treated plants caused a slight increase in apex size but had no effect on rate of

flowering (88). On the basis of these studies, Hussey (88) concluded that the

first two leaves competed strongly with the shoot apex for assimilates when

exposed to supraoptimal temperatures. Assimilate partitioning in the tomato

cultivars Roma VF and Saladette, high temperature sensitive and tolerant,

respectively, was reduced in response to exposure to heat stress conditions,

(380/250, day/night, for 48 hours) compared to plants exposed to control (260/150)

temperatures since carbon export from source leaves (61) and acropetal movement

and import of assimilates into young floral buds were reduced (60). Tomato

flower set is most sensitive to high temperatures 3 to 4 days after flower

initiation which apparently involves the sink demand of the inflorescence (60).

Moreover, high temperatures (350 to 400) were shown to decrease carbon fixation

to a greater extent in the heat-sensitive cultivar Roma VF compared to heat-

tolerant 'Saladette' (14).








22

Plant growth substances may regulate the responses to heat stress (92).

The primary response to heat stress may be an alteration in membrane integrity

causing phytohormone imbalances (92). However, it is not known whether

differences in hormonal activity represent the cause or an effect of heat

tolerance. The studies of high temperatures and cytokinin response constitute the

vast majority of evidence (119). Cytokinins synthesized in the roots have been

suggested to be "protective substances" in heat-stressed plants due to their

antioxidant activity and involvement with protein synthesis (112). Roots of wheat

(Triticum aestivum L. 'Svenno Varvete') died at temperatures of 35-360; however,

pretreatment with ethanol or kinetin protected the wheat roots against

supraoptimal temperature injury (167). In leaves of intact plants of the Christmas

begonia (Begonia X cheimantha Everett 'Prinsesse Astrid') cytokinin activity was

higher at 180 than at 210 or 240, as determined by bioassay, with a concomitant

decrease in adventitious bud formation at the higher temperatures (195). Using

bioassays, Cole (Ph.D dissertation cited by McDaniel [119]) observed inhibition of

cytokinin synthesis or translocation as a result of high root temperatures; and the

temperature tolerant 'Wando' pea maintained higher cytokinin activities in

response to high temperature exposure (400 for 9 days) than temperature sensitive

'Alaska'. Cytokinin, gibberellin, and ABA activity as determined by bioassays

were determined in stem xylem exudate from 'Inra 200' maize exposed to root

temperatures of 180, 230, 280 or 330 (9). Highest rate of cytokinin and

gibberellin export from the roots occurred at 280 and both decreased sharply with

increased temperature (330) exposure (9). Greatest rate of ABA export from the

roots occurred at 13-180 with decreases at higher or lower temperatures (9). A

heat treatment (47.50 for 2 minutes) to roots of 'Fronica' maize seedlings also

reduced endogenous cytokinin levels 6-fold as determined by a zeatin riboside-Ab








23

radioimmunoassay; and BA treatments to the corn seedlings reversed heat stress

inhibition of photosynthesis, chlorophyll accumulation, and chloroplast development

(25). Xylem (root) exudate from topped 'Great Northern' bean plants exposed to

460 or 47.50 for 2 minutes increased 4-fold in ABA content and decreased 6-fold

in cytokinin activity concomitant with changes in membrane permeability and ion

uptake (92). Sublethal high temperature treatments (460-490 for 2 minutes) to

roots and shoots of wild tobacco (Nicotiana rustica L.) increased ABA levels and

initially decreased cytokinin levels in leaf exudate similar to effects of water

stress (90). Pretreatment of tobacco seedlings with ABA or kinetin enhanced

recovery from heat shock (91), i.e., the effects of the high temperature treatment:

onset of senescence, inhibition of protein synthesis or promotion of proteolysis,

were reversed by kinetin applications (90). Levels of free ABA in shoots of

Venus' tomato seedlings exposed to low (150/100 or 100/50, day/night) or high

(350/250 or 450/350) temperatures were increased compared to levels in seedlings

exposed to optimal temperatures of 250/150 (51). No differences in leaf water

potentials were observed between the temperature treatments (51). Hence, the

increase in ABA levels in response to temperature stress may be involved in

enhancing plant temperature tolerance by modifying tissue water balance (51).

Auxin-like substance and auxin oxidasee" activities were higher at 280 than 200 in

chrysanthemum 'Sunbeam' exposed to short days (173). However, auxin and

gibberellin activities were reduced by exposure to high (380) temperatures for 5

hours in tomato floral buds compared to control temperatures (240-280/17-220,

day/night) (106).

The optimum temperature for ethylene production in plant tissues is

approximately 300 (186). Ethylene production is inhibited at temperatures above

350 in many fruits, and this inhibition is probably responsible for the problems









24

associated with fruit ripening at elevated temperatures (22). Ethylene production,

as determined by gas chromatography, was inhibited above 200 and 300 for tomato

and common apple (Malus domestic Borkh), respectively (118). Abnormal ripening

of avocado (Persea americana Mill. 'Hass') fruit at temperatures above 300 was the

result of a significant reduction in ethylene production due to increasing

temperatures (64). Avocado fruit maintained at 350 or 400 did not produce

appreciable quantities of ethylene and at 400 a climateric was not observed (64).

Increasing incubating temperatures from 250 to 350 increased ethylene production

twofold, but temperatures above 37.50 significantly reduced basal and wound

(stress-induced) ethylene production in leaf discs from 'Masterpiece' dwarf bean,

with complete inhibition at 450 and above (68).

The reaction rate of the conversion of ACC to ethylene was retarded by

temperatures above 290 in 'Golden Delicious' apple fruit tissue slices (8). Yu et

al. (186) demonstrated that high temperatures (350) primarily affected the

conversion of ACC to ethylene in 'Golden Delicious' apple plugs and auxin-treated

mungbean hypocotyls, and that synthesis of ACC was less sensitive to high

temperature inactivation. However, Horiuchi and Imaseki (84) observed that ACC

synthase activity induced by IAA was more sensitive to high temperatures (40

and above) than the conversion of ACC to ethylene in etiolated subapical

hypocotyl sections of mungbean. In addition, percent inhibition of ethylene

biosynthesis by rhizobitoxine, L-2-amino-4-(2-aminoethoxy)-trans-3-butenoic acid,

in common apple plugs increased with increasing temperatures (118).

Inhibition of ethylene biosynthesis by high temperatures may be caused by

pertubations in cellular membranes, i.e. alterations in conformation of membrane-

bound enzymes and/or membrane integrity. Stable, intact functional membranes

are requisite for ethylene biosynthesis (8,113). In subapical stem sections of








25

etiolated 'Alaska' pea seedlings, wound ethylene production increased from 240 to

360, with a dramatic decrease at temperatures above 360, indicative of a requisite

of membrane integrity for ethylene production (154). Membrane involvement is

also suggested from discontinuous Arrhenius plots of temperatures versus ethylene

production (118). Field (68) proposed that a loss in the integrity of the ethylene

production system was the result of temperatures above 37.50 as an increase in

electrolyte leakage correlated well with reductions in ethylene production. Field

(68) concluded that high temperatures altered membrane integrity and thus

adversely affected the activity of the membrane-bound enzymes of ethylene

synthesis. Furthermore, environmental effects on membrane function may also

involve de novo synthesis of ACC synthase (117).














CHAPTER III
HIGH TEMPERATURE EFFECTS ON FLORAL DEVELOPMENT AND
VEGETATIVE GROWTH OF CHRYSANTHEMUM X MORIFOLIUM

Introduction

High temperatures during production can delay flowering and induce

abnormal inflorescence development in chrysanthemums (144). This phenomenon is

commonly referred to as "heat delay" and has been shown to be induced by

temperatures in the range of 270 to 320C (27,31,40,144). The severity of heat

delay depends to a large extent on the tolerance or sensitivity of various

chrysanthemum cultivars to high temperatures.

Discrepancy exists in the literature on the effects of high temperatures

on chrysanthemum growth and floral development. Based on dates of visible bud

and flowering, Post and Lacey (144) reported that high temperatures did not

affect "bud initiation" (time from start of short days to visible bud), but delayed

"bud development" (time from visible bud to flower). Moreover, they observed

bract formation and a "large number" of developed florets on the capitulum in

response to high temperatures. Cathey (27) found that high temperatures

enhanced "bud initiation" but delayed "bud development." However, Cathey and

Borthwick (31) later reported that floral initiation was slightly delayed by

increased temperatures. Cockshull (40) has observed an increase in leaf

production and a delay in capitulum initiation in response to high temperatures;

however, these studies were conducted under continuous lighting which precluded

floret initiation. The objectives of the present study were to determine the most

sensitive stages) of floral development, to determine the effects of high








27

temperature on vegetative growth, and to investigate the effect of high-

temperature duration by comparing a sensitive to a tolerant cultivar.

Materials and Methods

Rooted chrysanthemum cuttings of 'Orange Bowl' (10-week-photoperiodic

response, high temperature sensitive) and 'Surf' (9-week-photoperiodic response,

high temperature tolerant) were planted in a soilless growth medium (Metro Mix

300, W.R. Grace Co., Cambridge, MA) in 225-ml styrofoam cups. Plants were

placed in a chamber (8.9 m2) in the Southeastern Plant Environment Laboratory

(phytotron) at North Carolina State University, Raleigh, NC, at a plant density of

86.5 plants m-2. Cool-white fluorescent and incandescent lamps provided a

photosynthetic photon flux (PPF) of approximately 640-650 pmol s-1 m-2 for 9 hr

per day. Plants were exposed to a 3-hour night interruption (2300-0200 hr) of

11-12 pmol s-1 m-2 from incandescent filament lamps (63). Two weeks after

planting, short-day photoinductive periods were initiated by providing a 15-hr

nyctoperiod from 1700 to 0800 HR. Ambient temperatures of 220.250C day and

180.250 night were maintained in one chamber, which served as the low

temperature or control chamber, and another chamber maintained at 300.250 day

and 260.250 night was used for the high temperature treatments. Temperatures

were monitored with a type T (copper-constantan) thermocouple in a shielded

aspirated housing (63). Temperatures selected for this study were based upon a

previous study conducted in the phytotron which involved exposing plants of both

cultivars at the start of short days to day/night temperatures of 180/140, 220/180,

260/220 and 300/260 until stage of flower maturation (unpublished data).

Plants were irrigated daily with 300 ml per plant of a modified Hoagland's

solution (7.6 mM nitrogen) and 300 ml deionized water (63). Plants were pinched

one week after planting and pruned to one lateral shoot when shoots were 3 cm








28
in length. Leaves below the pinch were pruned to four per plant for 'Orange

Bowl' and five per plant for 'Surf'. Plants were randomly divided into 15

subgroups (treatments) for 'Orange Bowl' and 11 treatments for 'Surf'. Treatments

consisted of exposing plants to high temperatures at the start of short days (week

1) or 2 (week 3), 4 (week 5), or 6 weeks (week 7) after the start of short days

for 2, 4, 6, 8, or 10 weeks (Table 3-1). Cultivars were in separate experiments

which were incomplete factorials in a randomized complete block design with

three replicates over time (three plantings 1 week apart) using six plants per

replicate/treatment/cultivar.

Leaf area, stem length, and leaf, stem and inflorescence dry weights of

the lateral shoot were recorded each week for two plants per treatment/replicate.

Number of short days to flower color (showing-color) and to when the outer rows

of florets were perpendicular to the pedicel (open-flower) were recorded.

Inflorescences were evaluated for abnormal development, floret color, and number

of florets at open flower.

At 3, 7, 11, 15, 19, 23, and 27 days after the start of short days, six

meristems from plants exposed to either low or high temperatures from the start

of short days were fixed in formalin-glacial acetic acid-95% ethanol (FAA) and

observed with a dissecting light microscope. Meristem diameter, appearance of

involucral bract primordia, number of rows of initiated florets, and number of

rows of florets with differentiated perianth were recorded. For electron

microscopy, selected meristems were dehydrated in a graded alcohol series, critical

point dried, coated with Au/Pd in a Technics Hummer V sputter coater, and

viewed with a Hitachi S-450 scanning electron microscope.









29

Table 3-1. Duration and timing of high temperature treatments that were used
on 'Orange Bowl' and 'Surf' chrysanthemums.


Number of short days after start of short days


Treatment 1 14 28 42 56 70


1-2 xxxxxxxxooooooo Y

1-4 XXXXXXXXXXooooooooooooooo

1-6 cccczcoz2oizpz

1-8 ) 3 xxxxx----------

1-10" xxxxx xxxxxxx xx xx x

3-2 xxxxxxxxxxxx

3-4 xxxxxxxxxxxxxxxxxxoooc

3-6

3-8x xc

5-2 xxxxxoooooooo

5-4 mxoooooooo0oooooooo0 c

5-6x

7-2 xxxxoocxxxxx

7-4x xxxxxxxxxx


ZFirst number refers to week at which high-temperature treatments were
initiated and the second number refers to length in weeks of high
temperature exposure.
YDuration in high temperature chamber.
X'Orange Bowl' only as 'Surf' is classified as a 9-week response cultivar and
'Orange Bowl' is classified as a 10-week response cultivar.








30
An additional study was conducted simultaneously at the Ornamental

Horticulture Department Greenhouses of the University of Florida, Gainesville, FL.

Cultivars and treatments were identical to the experiment previously described.

As the results obtained from this study were similar to those of the phytotron,

only the phytotron results will be reported.

Results and Discussion

Effect of High Temperature on Vegetative Growth

High temperature treatments initiated at the start of short days or at

week 3 (treatments 1-2, 1-4, 1-6, 1-8, 1-10, 3-2, 3-4, 3-6, and 3-8) increased leaf

and stem dry weights in 'Orange Bowl' compared to the control (Table 3-2). High

temperature treatments initiated at week 5 or 7 (treatments 5-2, 5-4, 5-6, 7-2,

and 7-4) did not substantially increase lateral shoot dry weights compared to the

control. At open flower, stem and leaf dry weights accounted for 51% and 74% of

the total lateral shoot dry weight for 'Orange Bowl' plants exposed to low and

high temperatures for the entire short day period (control and treatment 1-10),

respectively.

Stem and leaf dry weights of 'Surf' plants were greater when exposed to

high temperatures at the start of short days (treatments 1-2, 1-4, 1-6, and 1-8)

compared to plants exposed to high temperatures after week 3 or remaining at

low temperature until open flower (Table 3-3). Stem and leaf dry weights of

'Surf' plants exposed to low or high temperatures (control or treatment 1-8)

accounted for 46% and 62% of total lateral shoot dry weight, respectively. The

increase in shoot dry weight in both cultivars was attributable to an increase in

leaf and stem growth as temperature treatments had little effect on final

inflorescence dry weight (Tables 3-2 and 3-3).










Table 3-2. Effect of high- (300/260C) and low- (220/180) temperature treatments
during short days on leaf, stem, and inflorescence final dry weights of the lateral
shoot for'Orange Bowl' chrysanthemums.



Orange Bowl


Leaf dry Stem dry Inflorescence Total dry
weight weight dry weight weight
Treatment (g) (g) (g) (g)



1-2z 1.17 1.05 1.60 3.81

1-4 1.74 1.51 1.38 4.63

1-6 1.89 1.61 1.26 4.76

1-8 1.58 1.49 1.13 4.20

1-10 1.57 1.46 1.19 4.22

3-2 1.16 0.97 1.07 3.19

3-4 1.29 1.07 1.12 3.47

3-6 1.16 1.06 1.20 3.43

3-8 1.14 1.11 1.16 3.41

5-2 0.88 0.75 1.30 2.93

5-4 0.93 0.73 1.07 2.73

5-6 0.92 0.75 1.21 2.88

7-2 0.82 0.69 1.48 3.00

7-4 0.81 0.59 1.39 2.79

Control 0.76 0.66 1.37 2.78

Waller-Duncan 0.37 0.25 0.47 0.54
at 5% level


ZFirst number refers to week at beginning of which high-temperature treatments
were initiated and second number refers to length in weeks of high-temperature
exposure.










Table 3-3. Effect of high- (300/260C) and low- (220/180) temperature treatments
during short days on leaf, stem, and inflorescence final dry weights of the lateral
shoot for 'Surf' chrysanthemums.



Surf


Leaf dry Stem dry Inflorescence Total dry
weight weight dry weight weight
Treatment (g) (g) (g) (g)


1-2z 1.06 0.65 1.44 3.35

1-4 1.04 0.64 1.41 3.40

1-6 1.17 0.71 1.35 3.23

1-8 1.22 0.69 1.22 3.18

3-2 0.76 0.46 1.28 2.50

3-4 0.78 0.41 1.02 2.21

3-6 0.79 0.44 0.96 2.19

5-2 0.62 0.37 1.04 2.03

5-4 0.70 0.31 0.97 1.88

7-2 0.74 0.33 0.99 2.06

Control 0.65 0.36 1.19 2.22

Waller-Duncan 0.18 0.12 0.28 0.44
at 5% level


ZFirst number refers to week at beginning of which high-temperature treatments
were initiated and second number refers to length in weeks of high-temperature
exposure.








33

High temperatures caused an increase in total leaf area and stem length

as compared to low temperatures (Table 3-4). Increased leaf area was a result of

an increase in leaf size and number. Increase in stem length was a function of

increased node number as the mean internode length was not affected.

Effect of High Temperature on Floret Initiation and Differentiation

High temperatures delayed meristem transition to the reproductive state,

floret initiation, and floret differentiation in 'Orange Bowl'. Involucral bract

primordia, indicative of transition to the reproductive state, were evident at seven

and 11 days after the start of short days for 'Orange Bowl' plants exposed to low

and high temperatures, respectively. Statistical differences in meristem diameter

between the temperature treatments were not evident for 'Orange Bowl' plants

(data not shown). High temperatures decreased developmental rate from 1.7 rows

of florets (control) initiated per day to 0.8 rows per day (Figure 3-1A). Exposure

to high temperatures decreased the rate of perianth differentiation from 0.6 rows

of florets per day (control) to 0.3 rows per day (Figure 3-1B). Transition to the

reproductive state and rates of floret initiation and differentiation in 'Surf' were

not significantly affected by high temperature treatments (data not shown).

Effect of High Temperature on Flowering

High temperature treatments beginning with week 1 or 3 of short days

(treatments 1-4, 1-6, 1-8, 1-10, 3-2, 3-4, 3-6, and 3-8) increased the number of

short days to showing-color and to open-flower for plants of 'Orange Bowl' and

'Surf' (Figure 3-2). The most sensitive 2-week period was the third and fourth

weeks of short days. For treatments which provided only two weeks of high

temperature exposure (treatments 1-2, 3-2, 5-2, and 7-2), treatment 3-2 caused

the most delay. The amount of delay caused by treatment 1-4 was equaled by

treatment 3-2 which overlapped the last two weeks of the 1-4 treatment.










Table 3-4. Effect of high (300/260C) and low (220/180) temperatures during short
days on selected growth parameters of 'Orange Bowl' and 'Surf' chrysanthemums.



Number Leaf Internode Total Stem
of area per length leaf length
leaves leaf (cm) area (cm)
(SE) (cm2) (SE) (cm2) (SE)
Cultivar Treatment (SE) (SE)


Orange Bowl 300/260z 201 120.5 1.10.2 2454 22.61.0

220/180 161 90.5 1.00.1 1385 15.40.4

Surf 300/260 191 110.5 0.60.1 2149 12.00.5

220/180 161 90.5 0.70.1 1476 11.40.6


zDay/night temperatures.














A
(,
i- 25 18/22
0

20 -
w
F-
15 -

z 26/30

0
U,
0 5
cr

I 5 I I


B
LJ
a 10-
0
_J 18/22
u-
0
W 8
<---
z
w 6
w
LL
E 4 26/30

O
--
0


15 19 23 27
DAYS AFTER START OF SHORT DAYS




Figure 3-1. Effect of high (300/260C) and low (220/180) temperatures during
short days on number of rows of florets initiated (A) and number of rows of
florets with differentiated perianth (B) for 'Orange Bowl' chrysanthemums.
Vertical bars represent S.E. of the means.


























A C
e
de de


bc b


--c c





- e- -
eC






B D



de




e ee


Scd

a1b bb b b


12 14 1-6 1I8 HO 32 34 36 38 52 5-4 56 7-2 7-4 C 12 1 1-6 1-8 3-2 34 36
Control


TREATMENT






Figure 3-2. Effect of high- (300/260C) and low- (220/180) temperature treatments
during short days on time to stage of showing-color and open-flower for 'Orange
Bowl' and 'Surf' chrysanthemums. Bars with different letters are significantly
different at the 5% level according to Waller-Duncan Multiple Range Test. First
number of treatment designation refers to week at the beginning of which high-
temperature treatments were initiated and second number refers to length in
weeks of high-temperature exposure.


52 54 7.2


49

47

o 45

43

41

39



66


63

UL 60
0



5 54


51


'SURF'


'ORANGE BOWL'


ZControl








37
Flowering was not delayed in plants of either cultivar exposed to high

temperatures during the first two weeks of short days (treatment 1-2). Exposure

to high temperatures starting with week 5 of short days (treatments 5-2, 5-4, 5-6)

delayed flower opening in plants of 'Orange Bowl', but not of 'Surf'.

Cultivars differed in degree of developmental delay. 'Orange Bowl' plants

exposed to longest high-temperature-duration treatment (treatment 1-10) flowered

12 days later than plants maintained at low temperatures (Figure 3-2B). 'Surf'

plants exposed to high temperatures for the longest duration (treatment 1-8)

flowered 3 days later than plants maintained at low temperatures (Figure 3-2D).

Treatments which included high temperatures during the third and fourth

weeks of short days (treatments 1-4, 1-6, 1-8, 1-10, 3-2, 3-4, 3-6 and 3-8)

resulted in bract formation interior to the outer rows of florets of 'Orange Bowl'

plants. Noninvolucral bracts were evident and arrangement of outer rows of

florets was disorganized by the fourth week of short days (Figure 3-3). 'Orange

Bowl' plants exposed to high temperatures at the start of or at the third week of

short days until flower (treatments 1-10 and 3-8) formed bracteate buds; i.e., only

the outer rows of florets developed and the receptacle was covered with

noninvolucral bracts. Secondary inflorescences arising from individual florets

were also observed on plants forming bracteate buds (Figure 3-4). Increasing

duration of exposure to high temperature increased the degree of teratological

modifications. Concomitant with the increase in number of bracts was a decrease

in the number of florets per inflorescence. Number of florets per inflorescence

decreased with increasing duration of high temperature exposure (Figure 3-5). No

teratological modifications were noted on 'Surf' plants.

Floret color of 'Orange Bowl' plants was affected by exposure to high

temperatures after the seventh week of short days. Florets of plants exposed to



















































Figure 3-3. Meristem of'Orange Bowl' chrysanthemum exposed to high (300/260C)
temperatures for first 4 weeks of short days. Outer rows of florets are
disorganized and noninvolucral bracts are evident.




















































Figure 3-4. Secondary inflorescence formed on a receptacle of 'Orange Bowl'
chrysanthemum exposed to high temperatures (300/260C) from start of short days
until week 10.









40









x/ 0







r- .E .



I-- _._ 0
.. 0 a






Sx td s 0 0
3 c o
/ ry E. rj <

0 c





!- OD c -E
o ,- o::
(C) E-


x-- 3 ; M
0^ 0
(D z E










I I I
O 0 E
O O O
/i- U vi
ro <




00 00
o 0 0



S13dOl. JAO dH3gv N









41
high temperature at this time (treatments 1-8, 1-10, 3-6, 3-8, 5-4, 5-6, 7-2 and

7-4) were yellow (Royal Horticultural Society color group 12A) rather than the

normal orange-yellow (Royal Horticultural Society color group 14B) typical of the

cultivar. The observed fading of floret color tended to increase with increasing

duration of high-temperature exposure.

Our studies indicate that high temperatures during the short day

photoinductive period enhanced vegetative growth and retarded floral development.

The high temperature sensitive cultivar, 'Orange Bowl', was delayed in rate of

floret initiation and differentiation by high production temperatures. Moreover,

the supraoptimal temperatures used in this study perturbed floral development as

evident in the induction of anomalous bracts on the receptacle and the decrease

in the number of developed florets. The diminution in floret color may be

attributable to either a decrease in synthesis or an increase in degradation of

anthocyanins or carotenoids. The difference in tolerance or sensitivity to high

temperatures is relative since 'Surf' was delayed by high temperature treatments,

but not as severely as 'Orange Bowl', and abnormal development did not occur in

'Surf'.

Inhibition of floret initiation and/or development is a manifestation of

indirect heat injury as defined by Levitt (112). Specific chemical and/or physical

reactions may be inhibited or enhanced at supraoptimal temperatures. This form

of injury is usually reversible; however, the duration of the exposure to

supraoptimal temperatures is correlated with the length of time for development

to resume (112). Exposure to high temperatures (380) has been shown to alter

the endogenous auxin and gibberellin levels in tomato (Lycopersicon esculentum

Mill.) flowers and result in poor or inhibited fruit set (106). These high

temperature effects may be due to altered or inhibited assimilate transport (60).









42

Kinet (102) has suggested that the inhibition of inflorescence development may be

the result of competition for available assimilates between reproductive and

vegetative growth. The redistribution of assimilates may be affected by various

plant growth substances. It is thus hypothesized that the physical permutations

of normal inflorescence development and the enhancement of vegetative growth in

chrysanthemum may be attributed to pertubations in the normal balances of

endogenous plant growth substances as a result of high temperatures.














CHAPTER IV
INVOLVEMENT OF SUPRAOPTIMAL TEMPERATURES AND
PLANT GROWTH SUBSTANCES ON DEVELOPING
INFLORESCENCES OF CHRYSANTHEMUM X MORIFOLIUM

Introduction

High-temperature exposure (300/260, day/night) promoted vegetative

growth, delayed floret initiation and differentiation, and induced abnormal

inflorescence development in Chrysanthemum X morifolium Ramat. 'Orange Bowl'

plants (Chapter 3). The stages of inflorescence development most sensitive to

high temperatures were floret initiation and early differentiation. Inflorescence

development was unaffected by high temperatures after florets had differentiated

perianth. The presence of interphasic structures indicated that the reproductive

condition was unstable and the terminal regressed to the vegetative state.

Apparently, the temperature optimum for vegetative growth is higher than the

optimum for reproductive development in chrysanthemum (86).

Sub- or supraoptimal night temperatures reduced flower quality in

carnation, Dianthus caryophyllus L. 'White Sim', by increasing petal number,

resulting in a malformed flower referred to as a "bullhead" (73). The additional

petals were located on secondary growth centers in the case of suboptimal night

temperatures (50), but arose directly on the receptacles of plants exposed to

supraoptimal night temperatures (24.50). Exogenous applications of GA and/or IAA

to the developing apex during flower initiation promoted the development of

secondary growth centers with additional petals, while kinetin applications

increased the number of primary petals arising directly on the receptacle (73).

Since petal number and secondary growth center development were observed to be

43









44
effected by either temperature or plant growth substances, Garrod and Harris (73)

concluded that the effects of temperature might be mediated by alterations in the

endogenous balances or levels of plant growth substances.

Low-temperature treatment (120) prior to pistil and stamen differentiation

increased the incidence of bullhead flowers in 'Baccara' rose (Rosa sp. L.)

compared to higher, more optimal temperatures (18-240)(126). Similar to

carnations, bullhead is a malformation in rose in which the petals and petaloids

are smaller, distorted, and more numerous than normal flowers (126). Zieslin et

al. (196) found that bullhead flowers had lower gibberellin activity and higher

cytokinin activity than normal flowers. Injecting gibberellic acid into the

receptacles during low temperatures prevented malformations, and applications of

cytokinins at optimal temperatures resulted in the characteristic symptoms

expressed at suboptimal temperatures, i.e. a proliferation of adventitious florets

(196). Thus flower development is contingent upon a cytokinin-gibberrellin

balance during the stage of early flower development in rose (196).

Temperature influences the metabolic activity of plants (65). Supraoptimal

temperatures may limit assimilate supply, hinder translocation or utilization of

assimilates, and/or alter sink demand, thus inhibiting growth and development.

These responses are probably regulated by endogenous plant growth substances as

primary or secondary messengers of the environmental impingement. The

objective of this study was to determine the effects of applied plant growth

regulators on Chrysanthemum X morifolium 'Orange Bowl' exposed to supra- or

optimal temperatures and to quantify changes in selected endogenous plant growth

substances elicited by high temperatures.











Materials and Methods

Exogenously Applied Plant Growth Substances

Rooted cuttings of 'Orange Bowl' chrysanthemum were planted in a

soilless growth medium (Metro Mix 300, W.R. Grace Co., Cambridge, MA) in 12.5-

cm plastic pots. Plants were grown in a glass greenhouse providing a maximum

photosynthethic photon flux (PPF) of 1200 pmol s-1 m-2 and maintained at 3220

day and 2120C night temperatures. Noninductive photoperiods were provided for

2 weeks after planting. Plants were exposed to a 4-hour night interruption (2200-

0200 hr) of approximately 10 pmol s-1 m-2 from incandescent filament lamps.

Two weeks after planting, short-day photoinductive periods were initiated by

providing a 15-hr nyctoperiod from 1700 to 0800 HR. An identical glass

greenhouse was maintained at 3820 day and 2720 night temperatures.

Plants were fertilized weekly with 720 ppm N from a 20N-4.4P-16.6K

commercial soluble fertilizer and irrigated as needed. Plants were pinched to

seven leaves 1 week after planting and pruned to one lateral shoot when shoots

were 3 to 4 cm in length. Leaves below the pinch were pruned to five per plant.

At the start of the short-day photoinductive period, plants were randomly divided

and grown in either the 320/210 or 380/270 greenhouse until flowering.

Chemical treatments were applied when three to four rows of florets were

initiated as determined by dissection under a light microscope. This stage of

development was chosen because of its sensitivity to high temperatures (Chapter

3). Chemical treatments were applied 7 days later to plants in high-temperature

treatments than to plants in low-temperature treatments. A preliminary study

indicated that chemical applications during floret differentiation were ineffectual

(data not shown). Treatments were applied to the terminals of remaining plants

as droplets of 0.5 ml per plant using 1-ml syringes. The treatments were GA4,7









46
(GA4-[1a,2p,4aa,4bp,10p0]-2,4A-dihydoxy-1-methyl-8-methylene gibbane-1, 10-

dicarboxylic acid, 1,4A-lactone and GA7-[la,2p,4aa,4bp,10l?]-2,4A-dihydoxy-1-

methyl-8-methylene gibb-3-ene-1, 10-dicarboxylic acid, 1,4A-lactone) at 500, 1000,

or 2000 ppm; BA (6-N-benzylamino purine) at 75, 150, or 300 ppm; AVG (L-2-

amino-4-[2-amino-ethoxy]-trans-3-butanoic acid), aminoethoxyvinylglycine, at 125,

250, or 500 ppm; ABA (abscisic acid) at 250, 500, or 1000 ppm; daminozide

(butanedioic acid mono [2,2-dimethylhydrazide]) at 2500, 5000, or 10,000 ppm; and

ethephon (2-chloroethylphosphonic acid) at 1000, 2000, or 4000 ppm. Tween 20 at

a concentration of 0.05% was added to the chemical treatments and applied alone

as a control. Plants in each greenhouse were in randomized complete block

designs with six blocks per chemical treatment with one plant per block per

treatment; the study was repeated three times over two years with similar

treatments and environmental conditions. As the results obtained from these

three studies were similar, only the results from the final study are reported.

Number of short days to first flower color (showing-color) and to when

the outer rows of florets were perpendicular to the pedicel (open-flower) were

recorded. At stage of open-flower, number of leaves, length of the lateral shoot,

inflorescence diameter, number of florets, and number of noninvolucral bracts

were recorded.

Enzyme-Linked Immunosorbent Assay for Zeatin Riboside

Four replicates of 24 rooted cuttings each of 'Orange Bowl'

chrysanthemum were planted approximately 2 weeks apart and grown in glass

greenhouses maintained at 320/210 or 380/270 as described previously. All

cultural and environmental conditions were as previously described. Since the

most high-temperature-sensitive stages were floret initiation and early

differentiation (Chapter 3), terminal meristems from 10 randomly chosen plants








47
from each treatment per replicate were harvested when three to four rows of

florets were initiated and when three to six rows of florets had differentiated

perianth. Inflorescences from plants maintained under low temperatures were

harvested at stage of floret initiation 5 to 7 days prior to high-temperature-

treated plants and 5 to 6 days prior to high-temperature-treated plants at stage

of floret differentiation. After determination of fresh weight, terminal meristems

were ground in 80% ethanol, and extract was filtered, evaporated to dryness (350),

and re-extracted with water-saturated sec-butanol. Butanol layers were

evaporated to dryness and brought to volume with water. The sample was

adjusted to pH 3 to 4 with 2% NH4OH and stored at 4.

Linbro/Titertek EIA microtitration plates (Flow Laboratories, Inc.,

McLean, VA) were coated with monoclonal antibodies to zeatin riboside (Idetek,

Inc., San Bruno, CA), and samples were assayed using a modified procedure by

Eberle et al. (66). Wells of microtitration plates were filled with 200 1p of 250

pg/ml rabbit anti-mouse immunoglobulin (Sigma Chemical Co., St. Louis, MO) in 50

mM NaHCO3 buffer, pH 9.5, and incubated at 40 for 24 hr. Wells were washed

three times with a saline-Tween (0.5%) solution, pH 7.0, and then filled with 200

1p of monoclonal antibodies to zeatin riboside (100 /g/ml NaHCO3 buffer) and

incubated 24 hr at 4. Wells were washed as before, then filled with 200 pl 10

mg/ml rabbit serum albumin in 25 mM Tris-buffered saline, pH 7.5, incubated for

1 hr at 250 and then rewashed. Duplicate standards and samples of each

extraction were assayed. Immediately after the final wash, 100 pl of sample or

100 pl of trans-zeatin riboside (Sigma Chemical Co., St. Louis, MO) standard in

Tris-buffered saline with 0.1% gelatin (w/v) were added to the wells with 100 1p

of trans-zeatin riboside-alkaline phosphatase tracer (Idetek, Inc., San Bruno, CA)

in Tris-buffered saline and incubated for 3 hr at 4. To determine maximum and








48
nonspecific binding of the tracer, zero and excess (100 pmole/0.1 ml) of the

trans-zeatin riboside standards, respectively, were also assayed in duplicate.

Plates were washed as before with 0.5% saline-Tween solution. To determine

phosphatase activity, 200 pl of a p-nitrophenyl phosphate, PNPP, solution (1

mg/ml 0.9 M diethanolamine buffer, pH 9.8) were added to each well and

incubated for 1 hr at 370. To each well, 50 pl of 1.0 M NaOH was added to stop

the reaction. After 5 minutes, the optical density of each well was measured at

405 nm.

ACC Determination

Two replicates of 24 rooted cuttings each of 'Orange Bowl'

chrysanthemum were planted 3 weeks apart and grown in glass greenhouses

maintained at 320/210 and 380/270 as described previously. All cultural and

environmental conditions were as previously described except plants were not

pruned to one shoot per plant. Twenty-four terminal meristems from each

temperature treatment per replicate were randomly harvested when three to four

rows of florets were initiated, when three to six rows of florets had

differentiated perianth, and when all florets had differentiated perianth.

Inflorescences were harvested from low-temperature-treated plants 4 to 5 days

prior to high-temperature-treated plants at stage of floret initiation, 4 to 6 days

prior to high-temperature-treated plants at stage of floret differentiation, and 6

to 8 days prior to high-temperature-treated plants at stage of floret development.

Mersitems were placed in vials in liquid nitrogen and stored at -860 until analysis.

After determination of their fresh weight, ACC was extracted from the

terminal meristems and determined via a modification of the method of Lizada and

Yang (115). Frozen meristems were homogenized and extracted overnight at 40

with two to three times their weight in cold 5%-aqueous sulfosalicyclic acid. The









49

homogenate was centrifuged at 27,000g for 25 minutes. The acidified supernatant

was placed on a cation exchange resin (Rexyn 101 H') column (1 x 6 cm), washed,

and eluted with four column volumes of 2N NH40H. Eluate was evaporated until

dryness in vacuo at 380, reconstituted to two times the original fresh weight in

water, and assayed for ACC. To 500 pl of meristem extract in a 1.5-mi serum

reaction vial was added 1 pmol of HgCI2, and sufficient water to bring the final

volume to 900 jal. Vials were sealed with serum caps, and 100 ul of a 40 mixture

of 5.25% NaOCI and saturated NaOH (2:1, v/v) was injected. Vials were mixed on

a Vortex and held in ice for 5 minutes and remixed. A 1-mi gas sample from the

head space was injected into a gas chromatograph to determine ethylene content.

Extracts from each treatment per replicate for each stage of floret development

were assayed twice each with replicate internal standards. Amount of ACC

present in the samples was based on the determined conversion efficiency of the

internal standards.

Results

Exogenously Applied Plant Growth Substances

Number of leaves per lateral shoot was 201 on plants grown at 320/210,

day/night and 241 on plants grown at 380/270. Chemical treatments could not

affect the number of leaves within a temperature treatment, since time of

application florett initiation) was after leaf initiation had ceased. GA4,7

applications to plants exposed to either temperature treatment greatly increased

lateral stem length (Table 4-1). This increase was due to enhanced elongation of

the uppermost internodes. Application of the higher rates of ethephon (2000 and

4000 ppm) reduced lateral stem length on plants exposed to high or low

temperatures. Application of AVG, daminozide, or ABA at the highest rate (1000

ppm) reduced lateral stem length on plants exposed to low temperatures. Other










Table 4-1. Effect of low (320/210C) or high (380/270) temperatures during short
days and exogenously applied chemical treatments at stage of floret intiation on
lateral stem length and inflorescence diameter of 'Orange Bowl' chrysanthemums.



Treatment Lateral stem length Inflorescence
(cm) diameter (cm)
(SE) (SE)


320/210z 380/270 320/210 380/270


GA47
S500 ppm 41.31.2 60.21.6 11.80.6 9.21.2

1000 44.21.4 59.60.9 12.00.4 8.61.3
2000 43.92.1 65.82.5 11.50.6 10.00.0
Daminozide
2500 28.31.6 51.92.2 14.00.3 11.51.5
5000 28.01.2 48.81.5 13.70.5 10.30.9
10,000 25.21.0 47.32.0 13.80.4 11.50.6
BA
75 28.72.0 47.90.6 14.00.7 10.00.0
150 28.40.9 50.91.4 13.60.3 11.00.1
300 30.81.3 51.32.4 13.00.4 9.52.5
ABA
250 28.81.7 52.83.5 13.20.2 10.80.4
500 27.32.1 49.31.6 12.90.4 10.30.8
1000 26.70.5 49.81.7 12.80.5 11.00.5
Ethephon
1000 27.30.9 48.92.1 13.90.3 10.80.4
2000 25.40.7 45.61.6 13.60.4 9.20.3
4000 23.10.8 44.01.1 13.40.5 10.80.3
AVG
125 26.21.0 52.01.6 11.70.5 9.81.3
250 22.00.9 49.72.4 12.70.5 10.80.3
500 21.11.1 51.31.9 11.31.5 7.01.8
Control 28.60.9 51.32.8 13.50.2 10.00.9


zDay/night temperatures.








51
chemical treatments and rates did not affect lateral stem length. Nontreated

plants in high-temperature treatments were almost twice as tall as similar plants

in low-temperature treatments.

GA4,7 or AVG applied to plants exposed to low temperatures and the

highest rate of AVG (500 ppm) applied to high temperature-treated plants

decreased inflorescence diameter compared to nontreated plants (Table 4-1). No

chemical treatments increased inflorescence size. Inflorescence diameter of

nontreated plants in high-temperature treatment was 10.0 cm; and inflorescence

diameter of nontreated plants in low-temperature treatment was 13.5 cm.

Rate of inflorescence development was delayed by GA4,7, ethephon, and

AVG applications (Table 4-2). GA4,7 and ethephon treatments on plants exposed

to low-temperature treatments increased number of short days to stage of

showing-color 1 to 2 days, but did not effect number of short days to open-

flower. Plants exposed to high temperatures and treated with GA4,7 were delayed

3 to 6 days to stage of showing-color and 5 to 7 days to open-flower compared

to untreated plants. AVG treatments increased time to stage of showing-color

and open-flower on plants exposed to either temperature treatment. Other

chemical treatments did not affect rate of inflorescence development. Nontreated

plants in low-temperature treatments flowered before similar plants in high-

temperature treatments attained stage of showing-color.

The highest and lowest rates (500 and 2000 ppm) of GA4,7 increased the

number of florets on plants exposed to low temperatures, however, number of

florets on plants exposed to high temperatures were not affected by GA4,7

applications (Table 4-3). Number of noninvolucral bracts were increased by GA4,7

and BA applications on low- or high-temperature-treated plants. BA treatments

applied at the lowest rate (75 ppm) decreased the number of florets per











Table 4-2. Effect of low (320/210C) or high (380/270) temperatures during short
days and exogenously applied chemical treatments at stage of floret initiation on
number of short days to stage of showing-color and open-flower of 'Orange Bowl'
chrysanthemums.



Treatment Number of short Number of short
days to stage of days to stage of
showing-color open-flower
(SE) (SE)


320/210z 380/270 320/210 380/270


GA4,7
500 ppm

1000
2000
Daminozide
2500
5000
10,000


ABA


250
500
1000


Ethephon
1000
2000
4000
AVG


Control


390
401
400

371
381
391

381
380
391

380
381
381

400
401
391

410
410
441
380


601
590
621

571
581
582

561
551
552

571
561
561

571
581
561

591
601
621
561


500
501
501

491
491
511

491
500
501

501
491
501

511
511
501

521
510
531
500


780
761
781

721
733
701

712
722
702

712
701
702

743
752
724

740
732
750
712


ZDay/night temperatures.










Table 4-3. Effect of low (320/210C) or high (380/270) temperatures during short
days and exogenously applied chemical treatments at stage of floret initiation on
number of florets and noninvolucral bracts per inflorescence of 'Orange Bowl'
chrysanthemums.


Treatment Number of florets Number of noninvolucral
per inflorescence bracts per
(SE) inflorescence (SE)


320/210z 380/270 320/210 380/270


GA4,7
S500 ppm 39027 16512 7114 17326

1000 37337 2229 5520 16810
2000 36514 16226 8614 16923
Daminozide
2500 39412 27647 61 8729
5000 40912 2878 73 8711
10,000 45140 30115 95 997
BA
75 27426 1625 9823 1728
150 30522 12817 7515 21433
300 30837 16723 13227 18219
ABA
250 39142 22822 125 11119
500 38461 25432 104 15880
1000 37231 27637 174 11727
Ethephon
1000 3489 28322 10 609
2000 33632 25421 53 8920
4000 34210 28543 10 9116
AVG
125 43442 15527 208 16823
250 38227 16843 217 26333
500 24552 13120 4012 27445
Control 33119 21135 52 11429


zDay/night temperatures.









54

inflorescence on plants maintained at low temperatures, and BA applied at 75 or

150 ppm decreased the number of florets per inflorescence on plants maintained at

high temperatures. Daminozide treatments did not affect noninvolucral bract

number, but increased floret number on high or low temperature-treated plants.

ABA treatments did not have an effect on number of florets at either

temperature, however the highest rate of ABA (1000 ppm) increased the number of

noninvolucral bracts on plants exposed to low temperatures. Ethephon was the

only chemical treatment to reduce the number of noninvolucral bracts. Ethephon

applied at the lowest and highest rates (1000 and 4000 ppm) essentially eliminated

bract development on low-temperature-treated plants. On plants exposed to high

temperatures, ethephon at the lowest rate (1000 ppm) decreased the number of

bracts 47% compared to untreated high temperature plants, and floret number was

also increased. The highest rate of AVG (500 ppm) decreased floret number on

low- and high-temperature-treated plants. However, lower rates of AVG (125 and

250 ppm) increased floret number on plants exposed to low temperatures. AVG

treatments enhanced noninvolucral bract development on plants maintained at both

temperatures. Plants treated with GA4.7, BA, or AVG and maintained at high

temperatures had an equivalent or greater number of noninvolucral bracts

compared to number of florets per inflorescence. The terminal inflorescences of

these plants were considered bracteate buds, i.e., only the outer rows of florets

developed and the receptacles were covered with noninvolucral bracts. Nontreated

plants maintained under high-temperature conditions had 211 florets and 114

noninvolucral bracts per inflorescence. Nontreated plants maintained under low-

temperature conditions had 331 florets and five noninvolucral bracts per

inflorescence.










Enzyme-Linked Immunosorbent Assay for Zeatin Riboside

Endogenous zeatin riboside content increased with advancing stage of

inflorescence development and with exposure to high-temperature treatment (Table

4-4). Endogenous zeatin riboside content increased between stages of floret

initiation and differentiation 2.8 times and 2.1 times in inflorescences from plants

maintained under low and high temperatures, respectively. Endogenous zeatin

riboside content increased in inflorescences exposed to high-temperature treatment

at stages of floret initiation 2.3 times and at floret differentiation 1.7 times

compared to inflorescences exposed to low-temperature treatment.

ACC Determination

The level of ACC in the inflorescences was similar for plants exposed to

low or high temperatures at stage of floret initiation (Figure 4-1). During stage

of floret differentiation, a dramatic rise (4 times) in ACC content was observed in

low-temperature-treated inflorescences. ACC content of high-temperature-treated

inflorescences at stage of floret differentiation was not different from ACC

content at stage of floret initiation. ACC content in both low- and high-

temperature-treated inflorescences during stage of floret development were similar

and were approximately half of determined ACC levels in inflorescences at stage

of floret initiation.

Discussion

A primary response to heat stress may be an alteration in membrane

integrity causing phytohormone imbalances (92). Gibberellin and auxin activities

have been shown to decrease in response to high (380) temperatures in tomato

Lycopersicon esculentum Mill.) compared to low temperatures (240-280/17-220,

day/night) (106). However, our studies indicate that gibberellin levels, activity, or

sensitivity may increase in response to supraoptimal temperatures. GA4,7










Table 4-4. Effect of low (320/210C) or high (380/270) temperatures during short
days on endogenous zeatin riboside content of 'Orange Bowl' chrysanthemum
inflorescences harvested at development stages: 1) floret initiation (three to four
rows of florets initiated) and 2) floret differentiation (three to six rows of florets
with differentiated perianth).



Zeatin riboside
(pMol-g fr wt-') (SE)


Stage of inflorescence development 320/21oz 380/270


Floret initiation 23771 545180

Floret differentiation 66253 1140274


zDay/night temperatures.

















300

S380 / 270

250
L_
0)
200


a

0
0150



100



50-




FLORET FLORET FLORET
INITIATION DIFFERENTIATION DEVELOPMENT
STAGE OF
INFLORESCENCE DEVELOPMENT



Figure 4-1. Effect of low (320/210C) or high (380/270) temperatures during short
days on endogenous ACC content of 'Orange Bowl' chrysanthemum inflorescences
harvested at developmental stages: 1) floret initiation (three to four rows of
florets initiated), 2) floret differentiation (three to six rows of florets with
differentiated perianth), and 3) floret development (all florets with differentiated
perianth). Vertical bars represent SE of the means.









58
treatments were additive to high temperature treatments as applications to

initiated chrysanthemum inflorescences increased lateral stem length, decreased

inflorescence size, inhibited inflorescence development, and promoted bracteatebud

development. Although daminozide applications did not appreciably affect

chrysanthemum growth and development, floret arrangement of daminozide-treated

inflorescences grown under high temperatures resembled untreated inflorescences

in low-temperature treatments; exposure to supraoptimal temperatures distorts the

spiral arrangement of florets (Chapter 3).

Cytokinins synthesized in the roots were suggested to be "protective

substances" in heat-stressed plants (112). High temperature tolerance has been

correlated with increased cytokinin activities in pea (Pisum sativum L.) (119).

However, high temperature treatments decreased cytokinin activity in Phaseolus

vulgaris L. cv. Great Northern and Begonia X cheimantha Everett cv. Prinsesse

Astrid (92,195) and translocation in Zea mays L. cv. Inra 200 (9). BA treatments

to chrysanthemum inflorescences promoted bracteate bud formation of low and

high temperature-treated plants, and there was a marked increase in zeatin

riboside content in response to high temperature treatment. It is thus proposed

that gibberellin and/or cytokinin level, activity, or sensitivity increase in response

to supraoptimal temperatures in chrysanthemums causing abnormal inflorescence

development.

The optimum temperature for ethylene production in plant tissues is

approximately 300 (186). Ethylene production is inhibited at temperatures above

350 in many fruits, and this inhibition is probably responsible for inhibition of

fruit ripening at elevated temperatures (22). It appears that both ACC synthase

activity (84) and the conversion of ACC to ethylene (186) are affected by

supraoptimal temperatures. This inhibition of ethylene biosynthesis by high









59

temperatures may be caused by pertubations in cellular membranes, i.e. alterations

in conformation of membrane-bound enzymes and/or membrane integrity, as stable,

intact functional membranes are requisite for ethylene biosynthesis (8,113). In

this system, ethylene appears to be necessary for normal inflorescence

development, as AVG, a competitive inhibitor of ACC synthase, perturbed and

ethephon treatments promoted normal inflorescence development. AVG

applications reduced inflorescence size, retarded rate of inflorescence development,

and promoted bracteate bud formation. Ethephon treatments inhibited vegetative

growth and noninvolucral bract development on high-temperature-treated plants

and floret arrangement of ethephon-treated inflorescences at high temperatures

resembled untreated, low-temperature-treated inflorescences. The capacity for

ACC production is correlated with the ethylene production capacity of the tissue

(184,188). Thus the relatively high increase in ACC content on low-temperature-

treated inflorescences during floret differentiation may indicate an integral and

temporal role for ethylene in chrysanthemum inflorescence development.














CHAPTER V
SUMMARY AND CONCLUSIONS

High-temperature exposure (300/260, day/night) promoted vegetative

growth, delayed inflorescence initiation and differentiation, and induced abnormal

inflorescence development in Chrysanthemum X morifolium Ramat. 'Orange Bowl'

plants. The stages of inflorescence development most sensitive to high

temperatures were floret initiation and early differentiation. Inflorescence

development was unaffected by high temperatures after florets had differentiated

perianth. The presence of interphasic structures indicated that the reproductive

condition was unstable and the terminal regressed to the vegetative state.

A primary response to heat stress may be an alteration in membrane

integrity causing phytohormone imbalances (92). GA4,7 treatments were additive

to high-temperature treatments as applications to initiated chrysanthemum

inflorescences increased lateral stem length, decreased inflorescence size, inhibited

inflorescence development, and promoted bracteate bud development. Although

daminozide applications did not appreciably affect chrysanthemum growth and

development, floret arrangement of daminozide-treated inflorescences grown under

high temperatures resembled untreated inflorescences in low-temperature

treatments; exposure to supraoptimal temperatures distorts the spiral arrangement

of florets. BA treatments to chrysanthemum inflorescences promoted bracteate

bud formation of low and high temperature-treated plants, and there was a

marked increase in zeatin riboside content in response to high temperature

treatment. It is thus proposed that gibberellin and/or cytokinin level, activity, or

sensitivity increase in response to supraoptimal temperatures in chrysanthemums









61
causing abnormal inflorescence development. In this system, ethylene appears to

be necessary for normal inflorescence development, as AVG, a competitive

inhibitor of ACC synthase, perturbed and ethephon treatments promoted normal

inflorescence development. AVG applications reduced inflorescence size, retarded

rate of inflorescence development, and promoted bracteate bud formation.

Ethephon treatments inhibited vegetative growth and noninvolucral bract

development on high temperature-treated plants and floret arrangement of

ethephon-treated inflorescences at high temperatures resembled untreated, low-

temperature-treated inflorescences. The capacity for ACC production is correlated

with the ethylene production capacity of the tissue (184,188). Thus the relatively

high increase in ACC content on low-temperature-treated inflorescences during

floret differentiation may indicate an integral and temporal role for ethylene in

chrysanthemum inflorescence development.














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BIOGRAPHICAL SKETCH

Catherine Anne Whealy is a graduate of Fort Myers Senior High School and

a former employee of Yoder Brothers, Inc. She attended the University of the

South, Sewanee, Tennessee; New College, Sarasota, Florida; and received her

Bachelor of Science degree in horticultural science from North Carolina State

University in 1981. Whealy continued at North Carolina State University in the

graduate program in horticultural science and received her Master of Science in

1984.








I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.







Terkil A. Nell, Chairman
Professor of Horticultural Science


I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.






I .J, '77
/ames E. Barrett
Associate Professor of
Horticultural Science


I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.







JerKt M. Bennett
Associate Professor of Agronomy

I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.







R.'Hilton Biggs //
Professor of Horticultural Science








I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.







DeWayne t Ingram i
Professor df Horticultural Science





This dissertation was submitted to the Graduate Faculty of the College of Agriculture
and to the Graduate School and was accepted as partial fulfillment of the requirements
for the degree of Doctor of Philosophy.


August, 1987


Dean, Coll of Agriculture


Dean, Graduate School


















































UNIVERSITY OF FLORIDA


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