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This is to certify that the
dissertation entitled

THE INFLUENCE OF VERNALIZATION ON FLOWERING OF
CAMPANULA 'BIRCH HYBRID' AND DIANTHUS
GRATIANOPOLITANUS 'BATH'S PINK' AND THE REGULATION
OF FLOWERING OF COREOPSIS GRAND/FLORA 'SUNRAY' BY
VERNALIZATION, PHOTOPERIOD AND LIGHT QUANTITY

presented by

Sonali Ramesh Padhye

has been accepted towards fulﬁllment
of the requirements for the

PhD. degree in Horticulture

 

 

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2/05 p:/C|RC/DateDue.indd-p.1

THE INFLUENCE OF VERNALIZATION ON FLOWERING OF CAMPANULA
'BIRCH HYBRID’ AND DIANTHUS GRA TIANOPOLITANUS 'BATH'S PINK' AND
THE REGULATION OF FLOWERING OF COREOPSIS GRAND/FLORA
’SUNRAY‘ BY VERNALIZATION, PHOTOPERIOD AND LIGHT QUANTITY

By

Sonali Ramesh Padhye

A DISSERTATION
Submitted to
Michigan State University

In partial fulﬁllment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Horticulture

2006

ABSTRACT
THE INFLUENCE OF VERNALIZATION ON FLOWERING OF CAMPANULA
'BIRCH HYBRID' AND DIANTHUS GRA TIANOPOLITANUS 'BATH‘S PINK' AND
THE REGULATION OF FLOWERING OF COREOPSIS GRAND/FLORA
'SUNRAY' BY VERNALIZATION, PHOTOPERIOD AND LIGHT QUANTITY
By

Sonali Ramesh Padhye

Flowering of temperate herbaceous perennials is often regulated by
daylength (photoperiod) and low temperature (vernalization) to ensure
reproductive success. Greenhouse growers manipulate photoperiod and
vemalization to schedule ﬂowering of herbaceous perennials for speciﬁc market
dates. Our objective was to characterize the inﬂuence of vemalization on ﬂoral
evocation and subsequent ﬂowering characteristics of three herbaceous
perennials. We also investigated the effect of photoperiod and photosynthetic
daily light integral (DLI) on ﬂowering of Coreopsis grandiﬂora ‘Sunray’.

Following vemalization at -2.5 to 20 °C for O to 12 weeks, Campanula
'Birch Hybrid' exhibited a near-obligate vemalization requirement and all
ﬂowering responses studied were affected by the vemalization temperature,
duration and their interactions. The minimal and maximal cardinal temperatures
for vemalization were <0 °C and between 15 and 17.5 °C, respectively. The
optimal vemalization temperatures (Tom) depended on the flowering response
assessed and ranged between 0 to 12.5 °C for ﬂowering percentage to 5 to 7.5
°C for rate of progress to ﬂowering. Therefore, all relevant ﬂowering responses

should be considered when developing and interpreting vemalization models.

Dianthus gratianopolitanus 'Bath's Pink' exhibited a facultative
vemalization response to O to 10 °C and did not vernalize at 15 °C. Complete
ﬂowering was achieved following 24, 23 and 8 weeks at 0, 5 and 10 °C,
respectively. Vernalization temperature and duration affected time to anthesis,
and number of nodes and ﬂower buds and ﬂowers at anthesis. Based on the
minimum durations required to achieve maximum ﬂowering response, the order
of efﬁcacy of vernalizing temperature was 5 °C>0°C>>10°C.

Coreopsis grandiﬂora 'Sunray' exhibited a dual induction requirement for
ﬂoral evocation. The primary induction was fulﬁlled by either vemalization or
short days and secondary induction was stimulated by long days. Following sub-
optimal durations of primary induction treatment, vemalization was more effective
in promoting ﬂoral evocation than short days. DLl inﬂuenced ﬂowering
percentage, percent reproductive laterals, time to anthesis, number of

inﬂorescences, and plant height at anthesis.

ACKNOWLEDGEMENTS

I sincerely thank my advisor, Dr. Art Cameron for his guidance and
mentoring. I also thank Dr. Erik Runkle for his insights and help with
experimentation and Dr. Frank Telewski and Dr. Alan Prather for their helpful
suggestions. The encouragement of Dr. Royal Heins, statistical advice of Dr.
Bert Cregg and lab experience with Dr. Steve van Nocker is much appreciated. I
extend my appreciation to the supportive faculty and staff in the Department of
Horticulture. I am earnestly grateful to the supporters of MSU ﬂoriculture
program.

A special thank you to Mike Olrich, Anne Boone, Dave Joeright and
greenhouse undergraduate crew for help with plant care and their friendship. I
will always cherish the friendship of Marlene Ayala, Mauricio Canoles, Andrea
Beckwith, Mohamed Tawﬁk, Janelle Glady, Jessica DeGraaf and numerous
fellow graduate students. I appreciate the contagious smiles of Sandy Allen and
the affection of Cathy, Wayne and Laura Whitman. My friends in India and all
over the world, thanks for being there for me.

My parents, Mangala and Ramesh Padhye, my grandmother, Anandi
Joshi, my uncles, aunts, cousins and extended family in India, thank you for your
unconditional love, inspiration and support through every step of my life. Finally,
I am eternally grateful for the love of my husband, Narayanan Krishnan; you have

been a pillar of strength and my achievements are as much yours as mine.

TABLE OF CONTENTS

LIST OF TABLES ................................................................................................ vii
LIST OF FIGURES ............................................................................................. viii
CHAPTER I: VERNALIZATION: LITERATURE REVIEW ..................................... 1
Introduction ............................................................................................... 2
History and Deﬁnition of Vernalization ....................................................... 2
Ecological Signiﬁcance of Vernalization .................................................... 4
Relationship Between Vernalization and Acclimation ................................ 6
Vernalization Response Types .................................................................. 8
Perception of Vernalization ..................................................................... 11
Effective Thermoinductive Temperatures ................................................ 14
Quantitative Nature of Vernalization Response ....................................... 17
Memory of Vernalization .......................................................................... 18
Devernalization ........................................................................................ 1 9
Substitution of Vernalization Requirement .............................................. 20
Vernalization in Arabidopsis ................................................... . ................ 21
Vernalization in Wheat ............................................................................ 32
Future Perspectives ................................................................................ 34
Literature Cited ........................................................................................ 35

CHAPTER II: THE FLOWERING RESPONSE BEING ASSESSED REGULATES
THE OPTIMUM TEMPERATURE FOR VERNALIZING CAMPANULA ‘BIRCH

HYBRID’ ............................................................................................................. 43
Abstract ................................................................................................... 44
Introduction ............................................................................................. 45
Materials and Methods ............................................................................ 48
Results .................................................................................................... 52
Discussion ............................................................................................... 57
Literature Cited ........................................................................................ 65

CHAPTER III: DIANTHUS GRA TIANOPOLITANUS ‘BATH’S PINK’ HAS A

NEAR-OBLIGATE VERNALIZATION REQUIREMENT ...................................... 79
Abstract ................................................................................................... 80
Introduction ............................................................................................. 81
Materials and Methods ............................................................................ 83
Results and Discussion ........................................................................... 87
Literature Cited ........................................................................................ 92

CHAPTER IV: DAILY LIGHT INTEGRAL DURING SECONDARY INDUCTION
AFFECTS FLORAL EVOCATION IN COREOPSIS GRANDIFLORA 'SUNRAY'
FOLLOWING PRIMARY INDUCTION TREATMENTS OF VERNALIZATION OR

SHORT-DAY PHOTOPERIOD ........................................................................... 96
Abstract ................................................................................................... 97
Introduction ............................................................................................. 98
Materials and Methods .......................................................................... 100
Results .................................................................................................. 104
Discussion ............................................................................................. 108
Literature Cited ...................................................................................... 1 15

APPENDIX A: DAILY LIGHT INTEGRAL AFFECTS THE FLOWERING
RESPONSE OF COREOPSIS GRANDIFLORA 'SUNRAY' TO INDUCTIVE

TREATMENTS OF VERNALIZATION OR SHORT-DAY PHOTOPERIOD ...... 124
Introduction ........................................................................................... 125
Materials and Methods .......................................................................... 125
Results and Discussion ......................................................................... 129
Literature Cited ...................................................................................... 134

APPENDIX B: DAILY LIGHT INTEGRAL AFFECTS THE CRITICAL SHORT-
DAY AND LONG-DAY PHOTOPERIODS OF THE SHORT-LONG-DAY PLANT

COREOPSIS GRANDIFLORA 'SUNRAY' ........................................................ 138
Introduction ........................................................................................... 139
Materials and Methods .......................................................................... 139
Results and Discussion ......................................................................... 142
Literature Cited ...................................................................................... 146

vi

LIST OF TABLES

CHAPTER I
TABLE 1. Flower induction categories based on plant responses to vemalization
and long day photoperiod (adapted from Padhye et al., 2005) ........................ 10
CHAPTER II

Table 1. The minimum (Tmin), maximum (Tmax) and Optimum (T opt) cardinal
temperatures for vemalization of select herbaceous biennial and perennial
species .............................................................................................. 67

Table 2. Summary of optimum treatment temperatures for maximum ﬂowering
response at different durations for various ﬂowering responses assessed in
Campanula 'Birch Hybrid' ....................................................................... 68

Table 3. Summary of minimum treatment durations for maximum ﬂowering
response at different temperatures for various ﬂowering responses assessed in
Campanula 'Birch Hybrid' ....................................................................... 69

CHAPTER "I

Table 1. Planting date, average daily temperature (ADT) and average
photosynthetic daily light integral (DLI) of Dianthus gratianopolitanus “Bath’s Pink’
vernalized for different durations. ADT and DLl were calculated for each
ﬂowering plant from day of transplant to anthesis and for vegetative plants, 105 d
after transplant. MeantSE of ADT and DLI of 10, 30 and 240 plants are reported
for experiment 1, 2 and 3, respectively ...................................................... 93

CHAPTER IV

Table 1. Parameters of regression analyses (signiﬁcant at P=0.0005) relating to
ﬂowering percentage, rate of anthesis, time to anthesis, nodes at anthesis,
percent reproductive laterals and number of inﬂorescences at anthesis of
Coreopsis grandiﬂora 'Sunray’ following primary induction treatments of
vemalization or short days (SD) and secondary induction under high or low daily
light integrals (DLIs) ............................................................................. 118

vii

LIST OF FIGURES

CHAPTER I

Figure 1. Typical time-course of initiation and development of cold acclimation
and vemalization responses in Arabidopsis thaliana. Cold acclimation, leading to
freezing tolerance occurs within days after exposure to low temperature whereas
vemalization requires prolong exposure to low temperature (from Sung and
Amasino, 2005) ..................................................................................................... 7

Figure 2. Top: of Secale cereale 'Petkus' as a function of vemalization
temperature provided to imbibed seeds for 6 weeks (from Salisbury and Ross,
1992) ................................................................................................................... 1 5

Figure 3. The quantitative vemalization response of Petkus winter rye assessed
by measuring time to ﬂower computed after the end of therrnoinductive
treatments (adapted from Vince-Prue, 1975) ...................................................... 17

Figure 4. Pathways regulating transition from vegetative development to
ﬂowering in Arabidopsis thaliana (from Michaels and Amasino, 2000) ............... 22

Figure 5. Natural variability in ﬂowering time of Arabidopsis thaliana ecotypes.
(a) Summer annual types are naturally early ﬂowering and do not respond to
vemalization treatment, (b) ﬂowering of winter annual types is delayed without
vemalization treatment, (c) winter annual types ﬂower rapidly similar to summer
annuals following vemalization treatment (from Sung and Amasino, 2004a) ...... 23

Figure 6. Dose-dependent (rheostat-like) response of FLC in delaying ﬂowering
in Arabidopsis thaliana. In FRI background, ﬂowering is delayed based on the
number of FLC copies and transgenic plants with additional FLC copies behave
as biennials in absence of vemalization treatment (from Michaels and Amasino,
2000) ................................................................................................................... 25

Figure 7. Vernalization pathway regulated by FLC in Arabidopsis thaliana.
Vernalization induces VRN1, VRN2 and VIN3 genes which are repressors of FLC.
FLC in turn is a repressor of $001 and FT which are promoters of meristem

identity genes such as LFY and AP1 (from Sung and Amasino, 2005) .............. 30
Figure 8. Vernalization pathway mediated by VRN1 and VRN2 is responsible for
spring annual and winter annual growth habit in wheat ...................................... 33
CHAPTER II

Figure 1. Inﬂuence of temperature on node development of Campanula ‘Birch
Hybrid’ rooted cuttings. Open circles represent meantSE and solid lines
represent the regression equations generated using 722 observations from two
replications. The intercept and the slope of linear regression analysis for the rate
of node development were 00193100023 and 0.0066: 0.0002, respectively...70

viii

Figure 2. Flowering percentage of Campanula ‘Birch Hybrid’ as a function of
treatment temperature (A) and duration (B). Flowering percentage was
computed after growing plants in a glass greenhouse set at 20 °C. Plants
without open ﬂowers after 105 d in the 20 °C greenhouse were considered
vegetative. Averages of pooled data from two replications are presented ........ 71

Figure 3. Inﬂuence of temperature treatment and its duration on rate of progress
to ﬂowering (A and B), time to ﬂower (C and D) and total time to ﬂower (E and F)
of Campanula ‘Birch Hybrid’. Rate of progress to ﬂowering was computed as
1/days to ﬁrst open ﬂower from end of temperature treatment. Time to ﬂower
was computed starting from end of temperature treatment and total time to ﬂower
was computed starting at the beginning of temperature treatments. MeanstSE
data pooled from two replications are presented ......................................... 72

Figure 4. Inﬂuence of temperature treatment (A) and its duration (B) on thermal
time to ﬂower from beginning of treatment. Thermal time was calculated as
average daily temperature minus estimated leaf unfolding base temperature of
-2.9 °C and expressed in °C d. Means:I:SE of data pooled from two replications
are presented ...................................................................................... 74

Figure 5. Percent reproductive laterals one week after ﬁrst open ﬂower of
Campanula ‘Birch Hybrid’ as a function of treatment temperature (A) and duration
(B). MeansiSE (A) and means (B) of 18 observations pooled from two
replications are presented ...................................................................... 75

Figure 6. Inﬂuence of vemalization temperature on number of open ﬂowers
counted one week following ﬁrst open ﬂower in Campanula ‘Birch Hybrid'
vernalized for 3 weeks (A), 5 weeks (B), 7 weeks (C), 9 weeks (D) and 12 weeks
(E). Squares represent meaniSE in replication 1 and diamonds represent
meaniSE in replication 2 ....................................................................... 76

Figure 7. Number of ﬂower buds and ﬂowers on 4‘", 5th and 6th plant to ﬂower
after ﬁrst open ﬂower, counted one week following the ﬁrst open ﬂower on
Campanula ‘Birch Hybrid’ plants as a function of (A) treatment temperature and
(B) treatment duration. Means:I:SE (A) and means (B) of 3 observations are
presented. Treatments resulting in <4 ﬂowering plants were considered to have
0 ﬂower buds and ﬂowers ...................................................................... 77

Figure 8. Thermal time to ﬂower from 5 weeks (A) and 7 weeks (B) after
beginning of temperature treatment as a function of treatment temperature.
Thermal time was calculated 5 or 7 weeks from the beginning of temperature
treatment to the day of ﬁrst open ﬂower as average daily temperature minus
estimated leaf unfolding base temperature of -2.9 °C and expressed in °C d.
MeanSiSE of data pooled from two replications are presented for treatment
combinations resulting in >70% ﬂowering .................................................. 78

CHAPTER III

Figure 1. Flowering response of Dianthus gratianopolitanus “Bath’s Pink’ in
experiment I following vemalization at 5 °C at increasing durations. Open
symbols represent the averages for ﬂowering plants and vertical bars represent
standard errors. In A, ﬂowering percentage was computed 105 d after
transplant ........................................................................................... 94

Figure 2. Flowering response of Dianthus gratianopolitanus ‘Bath’s Pink’ in
experiment II and Ill following vemalization at 0, 5, 10 or 15 °C at increasing
durations. Open symbols represent the averages for ﬂowering plants and vertical
bars represent standard errors. In A, ﬂowering percentage was computed 105 d
after transplant. In D and E, data were collected at anthesis ......................... 95

CHAPTER IV

Figure 1. Flowering of Coreopsis grandiﬂora 'Sunray' in response to primary
induction treatment of vemalization (O/EI) at 5 °C with 16-h long days (A-D) or
short-days (SD; A/V) under 9-h photoperiod at 20 °C (E-H) for varying durations
and secondary induction treatments of long days under high (OIA) or low (El/V)
daily light integrals (DLI). Flowering response was assessed as ﬂowering
percentage 105 d from initiation of secondary induction (A, E), rate of progress to
ﬂowering computed as reciprocal of time to anthesis with ﬂowering rate of
vegetative plants reported as zero (B, F), time to anthesis from initiation of
secondary induction (C, G) and number of nodes from initiation of secondary
induction until anthesis (D, .H). Closed symbols are based on 10 observations
and open symbols represent averageiSE of 10 replicates for B, F and
averageztSE of ﬂowering plants for C-D, G-H. Solid and dashed lines represent
regression analyses for individual plants under high and low DLls,

respectively ..................................................................................... 1 19

Figure 2. Correlation analysis between time to anthesis and time to visible
inﬂorescence of Coreopsis grandiﬂora 'Sunray' plants computed from the start of
secondary induction to anthesis and appearance of ﬁrst visible inﬂorescence,
respectively (A). Correlation analysis between time to anthesis and number of
nodes developed from the start of secondary induction to anthesis (B). Data are
pooled for all ﬂowering plants that were previously given primary induction
treatments of vemalization (16-h photoperiod at 5 °C) or short-days (9-h
photoperiod at 20 °C). Each regression line is generated usin over 225
observations for all ﬂowering plants (signiﬁcant at P=0.0001; =0.96 and 0.79 for
A and B, respectively) .......................................................................... 121

Figure 3. Flowering characteristics of Coreopsis grandiﬂora 'Sunray' in response
to primary induction treatment of vemalization (O/D) at 5 °C with 16-h long days
(AD) or short-days (SD; A/V) under 9-h photoperiod at 20 °C (E-H) at varying
durations and secondary induction treatments of long days under high (OIA) or
low (DIV) daily light integrals (DLI). Flowering characteristics assessed included
total laterals (A, E), percent reproductive laterals (B, F), number of inﬂorescences

(C, G) and plant height (D, H) at anthesis. Plant height was measured from plant
base to the highest point. Open symbols represent averagetSE of 10 replicates
for 8-0, F-G and averageiSE of ﬂowering plants for A, D, E, H. Solid and
dashed lines represent regression analyses for individual plants under high and
low DLls, respectively ......................................................................... 122

APPENDIX A

Figure 1. Average daily temperature (ADT; A, C) and daily light integral (DLI; B,
D) of Coreopsis grandiﬂora 'Sunray' plants forced at a 20 °C greenhouse
temperature setpoint and a 16-h photoperiod. Prior to forcing, plants were
provided inductive treatments for varying durations either by vernalizing at 5 °C
under 16-h photoperiod in 72-cell trays (A, B) or by growing under SD (9-h
photoperiod) at 20 °C in 13-cm square pots (C, D). Diamonds represent data
from experiment I and squares represent data from experiment II. Each symbol
represents ADT or average DLI for an individual plant and was calculated from
transplant until anthesis and 105 d from transplant for ﬂowering and vegetative
plants, respectively. Error bars representing SE not presented since smaller than
the symbol size .................................................................................. 135

Figure 2. The inﬂuence of vemalization treatment (A-F) or SD treatment (G-L) on
ﬂowering percentage (A, G), time to visible inﬂorescence (B, H), time to anthesis
(C-l), number of inﬂorescences (D-J), number of lateral shoots (E, K) and plant
height (F, L) of Coreopsis grandiﬂora 'Sunray'. Plants were vernalized at 5 °C in
72-cell trays and exposed to short day (SD) treatment (9-h photoperiod at 20 °C)
in 13-cm square pots prior to forcing under a 16-h photoperiod at 20 °C.
Flowering percentage was computed 105 d after transplant. D-F and J-L were
counted on the day of anthesis. Diamonds represent data from experiment I and
squares represent data from experiment ll. MeaniSE of 9 and 10 replicates are
presented for experiment I and II, respectively .......................................... 136

APPENDIX B

Figure 1. Average daily temperature (ADT; A) and average daily light integral
(DLI; B) of Coreopsis grandiﬂora 'Sunray' plants grown in a 20 °C greenhouse.
ADT and average DLI were computed from the beginning of the inductive
treatments until anthesis and 105 d from initiation of forcing for ﬂowering and
vegetative plants, respectively. Dark lines represent the data from experiment I
and grey lines represent data from experiment II ....................................... 147

Figure 2. Flowering responses of Coreopsis grandiﬂora 'Sunray' plants following
critical SD photoperiod of 10 to 14 h (A-F), critical LD photoperiod of 12 to 15 h
(G-L) and constant photoperiod of 11 to 15 h (H-R). Following critical SD
treatment for 3 weeks, all plants were forced under a 16-h photoperiod and prior
to critical LD treatment, all plants were treated with a 9-h photoperiod for 3 weeks.
The inﬂuence of photoperiod treatments on ﬂowering of Coreopsis grandiﬂora
'Sunray' was assessed based on ﬂowering percentage (A, G, M), time to visible
inﬂorescence (B, H, N), time to anthesis (C, I, 0), number of nodes at anthesis (D,

xi

J, P), number of ﬂowering lateral shoots (E, K, O) and number of inﬂorescences
(F, L, R). Flowering percentage was computed 105 d after initiation of force. D-F,
J-L and P-R were counted on the day of anthesis. Diamonds represent data

from experiment I and squares represent data from experiment ll. MeantSE of 8
replicates are presented ....................................................................... 148

xii

Chapter I

Vernalization: Literature Review

Vernalization: Literature Review

Introduction

In the life cycle of a plant, the transition from vegetative to reproductive
phases of development can determine the success of ﬂower development,
pollination and timing of seed dispersal. Therefore, in most plants, this transition
to ﬂowering is tightly regulated by various endogenous and exogenous factors to
ensure reproductive success (Amasino, 1996; Amasino, 2005). The endogenous
factors affecting ﬂowering consist of developmental age, availability of
assimilates and plant hormones (Bemier, 1986). The exogenous factors
affecting ﬂowering include environmental cues such as light quality and quantity,
stresses such as nutrient deﬁciency, high temperature and water availability and
two primary cues, daylength and vemalization (Fausey, et al., 2001; Mouradov et
al., 2002; Simpson and Dean, 2002; Sung and Amasino, 2004a; Yanovsky and
Kay, 2003). Together, the factors affecting ﬂowering regulate the ﬂowering time
of plants to ascertain the completion of ﬂowering and seed development during

favorable environmental conditions (Amasino, 1996).

History and Deﬁnition of Vernalization

It has long been known that winter cereals needed to be planted prior to
the end of winter in order to ensure ﬂowering within a year, whereas spring
cereals ﬂowered readily after planting in the spring (Chouard, 1960). In 1857,
Klippart researched environmental factors responsible for ﬂowering of winter

cereals and showed that it was exposure to cold that induced winter cereals to

ﬂower following return to warm temperature (reviewed by Chouard, 1960). In
1898, von Seelhorst and, Gassner, in 1918, extensively studied the winter and
spring forms of various species. Gassner discovered the ability of vernalizing
imbibed seeds followed by Lysenco’s ﬁnding that partially imbibed seeds can be
vernalized before being mechanically sowed (reviewed by Chouard, 1960).
Since spring cereals are called yarovoe in Russian (Chouard, 1960), Lysenco
coined the term “yarovizatzya” in Russian to describe conversion of winter
cereals into spring cereals by cold (Thomas and Vince-Prue, 1984). Vemum
means spring in Latin (Chouard, 1960; Lang 1965). Therefore, vemalization, the
Latin translation of “yarovizatzya” essentially means “springization” or conversion
of winter annuals into spring annuals (Salisbury and Ross, 1992). The term
vemalization has since commonly been used in scientiﬁc literature.

Vernalization has been deﬁned as “the process whereby ﬂowering is
promoted by a cold treatment given to a fully hydrated seed or to a growing plant”
(Amasino, 2002). Additionally, the low temperature treatment intended to induce
or promote ﬂowering is also referred to as vemalization treatment (Napp—Zinn,
1987). Vernalization promotes ﬂoral evocation, though the actual development of
ﬂoral primordia generally occurs only following return to higher temperatures
(Vince-Prue, 1975). Thus, during vemalization there is a temporal separation
between the perception of low temperature and the ﬂowering response. For
several woody plants, including lilac, exposure to low temperature promotes
appearance of ﬂowers by breaking dormancy. Hence, in these woody plants the

role of low temperature is promotion of growth in the pre-formed buds and not the

promotion of ﬂoral evocation, indicating that vemalization and dormancy are two
distinct phenomena (Chouard, 1960). Interestingly, vemalization and dormancy
share some common physiological characteristics such as the effective treatment
temperatures and durations, non-transmissibility of the perceived signal, site of
low temperature perception and the suggested requisite of mitotic activity
(Metzger, 1996). Based on above similarities between vemalization and
dormancy, Metzger (1996) postulated that vemalization and dormancy may have
common molecular mechanisms, however at present there is no supporting

molecular evidence available (Sung and Amasino, 2005).

Ecological Signiﬁcance of Vernalization

The process of sexual reproduction in plants consisting of ﬂowering and
seed set is energy-expensive (Sachs, 1987) and therefore, its scheduling in
appropriate environmental conditions is essential for the survival of plants. A
vemalization requirement imposes that plants remain vegetative in autumn and
winter and ﬂower only during the spring or summer following exposure to cold for
an adequate duration (Simpson and Dean, 2002). Thus, a vemalization
requirement is ecologically advantageous to plants native to the temperate
climates with severe winters since it facilitates programming ﬂowering during
favorable environmental conditions by “perceiving” seasons (Sung and Amasino,
2004a) and enhances the probability of completion of the plant life cycle and
seed set (Sheldon et al., 2000a; Henderson et al., 2003). The life cycle of

annuals ends following ﬂowering and seed set therefore, the timing of ﬂowering

in annuals is especially important, making vemalization requirement of particular
signiﬁcance in the life cycle of annuals (Amasino, 2003).

Arabidopsis thaliana (L.) Heynh. ecotypes are native to diverse
geographical conditions and exhibit natural variation in ﬂowering time and
vemalization responses. Therefore, Arabidopsis ecotypes are excellent
candidates to study the correlation between their geographical origins and
responsiveness to vemalization. Following evaluation of 40 Arabidopsis
ecotypes, Johanson et al. (2000) reported that most late ﬂowering ecotypes that
responded to vemalization were from the northern latitudes while most early
ﬂowering ecotypes that did not respond to vemalization originated in central and
eastern Europe where winters are comparatively mild. However, Johanson et al.
(2000) did not ﬁnd a correlation between ﬂowering time and mean temperatures
or altitude in the ecotypes studied. Although favorable seasonal cycles may
have had a signiﬁcant impact on evolution of mechanisms regulating ﬂowering
time such as vemalization, variable ﬂowering times may also be advantageous in
other environments including locations with severe winters preventing seed
germination, those that support multiple generations in a year (Johanson et al.
2000), locations where plant populations may be affected by agricultural activities
such as harvest or tillage or succession or environmental stresses such as
drought (Mitchel-Olds, 1996). Therefore, evolution of vemalization requirement
probably depended not only on the geographical origin, but different
microenvironments may also have had a great impact on its evolution,

suggesting the complexity of the evolutionary process (Johanson et al., 2000).

A vemalization requirement is absolute in crops such as Beta vulgaris L.
(sugar beet); without vemalization, plants remain vegetative for years (Salisbury
and Ross, 1992). In contrast, when clonally propagated vemalization-requiring
herbaceous perennial species such as Oenothera fruticosa L. 'Youngii-lapsley'
(narrowleaf evening-primrose) are grown without vemalization for a prolonged
period of time, a few plants in the population do ﬂower (Clough et al., 2001). This
non-uniform vemalization requirement among the plant population may be
advantageous since at least a few plants will complete ﬂowering in the absence

of a vemalization treatment.

Relationship Between Vernalization and Acclimation

Cold acclimation is another plant response observed following exposure to
low temperature. Cold acclimation occurs following exposure to cold but non-
freezing temperatures and leads to freezing tolerance in plants of temperate
origins (Thomashow, 1999). Freezing tolerance is essential for survival of plants
where ambient temperatures are below freezing in the winter. Due to common
occurrences of sudden decreases in ambient temperatures in autumn and winter
in temperate climates, it is imperative that plants acclimate to cold quickly. On
the contrary, longer durations of low temperature exposure are needed to meet
vemalization requirement presumably since ﬂowering in response to a transient
decrease in temperatures would negate the ecological beneﬁt of having a
vemalization requirement (reviewed by Sung and Amasino, 2005). Raphanus

sativus L. 'Chinese Radish Jumbo Scarlet' (radish) is an exception to

vemalization in response to long exposures to thermoinductive temperatures as it
vernalizes following 8 d exposure to 6 °C (EnNin et al., 2002). Arabidopsis on the
other hand vernalizes in response to longer durations at thermoinductive
temperatures. The overall differences in the time of initiation and development of
cold acclimation and vemalization response following exposure to low

temperature in Arabidopsis are illustrated in Fig. 1.

 

 

 

 

1

_ (Lair; Act: :lmatim Vernalization
:

O

n‘i

m

l)
a:
0%ﬁIfIIIFIIIT‘L‘I r r r
012345678910 20 30 40 50

Time in cold (days)
Figure 1. Typical time-course of initiation and development of cold acclimation
and vemalization responses in Arabidopsis thaliana. Cold acclimation, leading to
freezing tolerance occurs within days after exposure to low temperature whereas
vemalization requires prolong exposure to low temperature (from Sung and
Amasino, 2005).
The molecular mechanism involved in induction of cold acclimation in

Arabidopsis involves a cascade of cold-induced transcription regulators known as

C-repeat binding factors (CBFs), resulting in the induction of proteins that provide

protection from cold (Thomashow, 1999). In addition, phytohonnone abscisic
acid (ABA) has been known to be associated with cold acclimation responses.
The vemalization response of Arabidopsis was not affected following
overexpression of CBF, impairing ABA biosynthesis by mutagenesis or elevation
of ABA levels by exogenous application or water stress (Liu et al., 2002). Hence,
cold acclimation and vemalization responses are mediated through separate
pathways in Arabidopsis. However, it is likely that the two pathways share some
common components at upstream level, particularly in early perception of cold

(reviewed by Sung and Amasino, 2005).

Vernalization Response Types

Based on their vemalization responses, plants can be grouped into
obligate (qualitative) and facultative (quantitative) response categories (Lang,
1965). Plants with an obligate vemalization response acquire the ability to ﬂower
following exposure to vemalization treatment. For example, Campanula 'Birch
Hybrid' plants did not ﬂower without exposure to cold and therefore, their
ﬂowering response was categorized as obligate (Enﬁeld, 2002; Niu et al., 2004).
In plants exhibiting a facultative vemalization response, vemalization accelerates
ﬂowering and/or improves ﬂowering characteristics including increasingthe
percentage of plants ﬂowering in the population, synchronization of ﬂowering and
increasing ﬂower number. For example, ﬂowering of Osteospennum ecklonis
(DC.) Nor. (Vanstaden's river daisy) plants was hastened and synchronized

following vemalization treatment and vernalized plants had more buds than non-

vernalized plants (Suzuki and Metzger, 2001). It is noteworthy that often plants
are categorized as obligate vemalization responsive based on them remaining
vegetative for a certain period of growing time following vemalization treatment.
Additional growth at warm temperatures may result in ﬂowering of some plants in
the non-vernalized population, indicating that botanically, the vemalization
response of such species is facultative. However, horticulturally such ﬂowering
response may still be considered obligate (Runkle et al., 2001). Clough et al.
(2001) categorized the vemalization requirement of Oenothera fruticosa 'Youngii-
lapsley' as horticulturally obligate, since, in this species, ﬂowering was rare
(~0.6%) and delayed without a vemalization treatment.

Flowering in Response to Vernalization and Photoperiod. Similar to
vemalization, plant responses to photoperiod can also be categorized as obligate
and facultative and many plants have a dual regulation of ﬂowering based on
their response to vemalization and photoperiod (Vince-Prue, 1975). Typically,
plants responding to both vemalization and photoperiod treatment are long day
(LD) plants however, Chrysanthemum xmon'folium Ramat. (ﬂorist's daisy) is an
exception and has been known to respond to vemalization and short day (SD)
photoperiods (Vince-Prue, 1975). Categorization of plants based on their

ﬂowering responses to vemalization and LD photoperiod is presented in Table 1.

Table 1. Flower induction categories based on plant responses to vemalization

and long day photoperiod (adapted from Padhye et al., 2005).

 

 

 

 

 

 

Long day Vernalization response

photoperiod

response None Facultative Obligate

None Vernalization Vernalization Vernalization
treatment or LD treatment is not treatment is required
photoperiod do not required for ﬂowering for ﬂowering.
affect ﬂowering. but improves ﬂowering Plants are day-neutral

characteristics such after vemalization.
as synchronization of
ﬂowering, reducing
time to ﬂower and/or
improves plant quality
characteristics such
as number of ﬂowers
and number of lateral
shoots produced.
Plants are day-neutral
after vemalization.

Facultative Vernalization Both vemalization Vernalization
treatment is not treatment and LD treatment is absolutely
required for ﬂowering. photoperiod are not required for ﬂowering.
Flowering required for ﬂowering LD photoperiod
characteristics are but improve ﬂowering improves ﬂowering
improved by LD characteristics. characteristics.
photoperiod.

Obligate Vernalization is not Vernalization Vernalization

 

required for ﬂowering.
LD photoperiod is
required for ﬂowering.

 

treatment is not
required for ﬂowering
but improves ﬂowering
characteristics.

LD photOperiod is
absolutely required for

 

ﬂowering.

treatment and LD
photoperiod are
absolutely required for
ﬂowering

 

Following vemalization, the photoperiodic response of some species is

altered. For example, non-vernalized Leucanthemum xsuperbum (Bergmans ex

J. W. Ingram) D. H. Kent 'Snowcap' (Shasta daisy) and Lobe/fa xspeciosa

'Compliment Scarlet' (hybrid cardinal ﬂower) required LD for ﬂowering however,

following vemalization the plants responded facultatively to LD (Runkle et al.,

1998; Runkle, 1999a). In addition, vemalization can alter the critical photoperiod

for ﬂowering as reported in the case of Rudbeckia fulgida Ait. 'Goldstrum' (orange

10

 

coneﬂower). In this Rudbeckia cultivar, following vemalization treatment, the
critical photoperiod shifted from 14 to 13 h (Runkle et al., 1999b). In Arabidopsis,
the photoperiod pathway is distinct from the vemalization pathway and both
these pathways integrate downstream at genes involved in induction of ﬂoral
evocation. However, this does not explain the mechanisms involved in altering
the photoperiodic response following vemalization reported in many herbaceous

perennials.

Perception of Vernalization

Prerequisites for Vernalization. Wellensiek (1962, 1964) illustrated that
vernalized growing leaves and roots of Lunan'a annua L. (moneyplant)
regenerated into ﬂowering shoots whereas, leaves fully grown prior to
vemalization treatment regenerated into vegetative shoots. Therefore, it was
inferred that mitotic activity is a prerequisite for vemalization (Wellensiek, 1964).
Contrasting results were reported by Metzger (1988) when similar regeneration
experiments were performed with Thlaspi arvense L. (ﬁeld pennycress). Mature
leaves excised from vernalized Thlaspi plants regenerated in ﬂowering plants,
thereby questioning the prerequisite of mitotic division for the perception of
vemalization. Metzger (1988) postulated that mitotic activity may not be required
for perceiving vemalization but it may be necessary for the perpetuation of the
vernalized state. Although the possibility of localized mitosis existed in fully
expanded Thlaspi leaves, it was later postulated that DNA replication may still

occur in those leaves (Michaels and Amasino, 2000). Since DNA replication

11

often plays a role in reprogramming gene expression, Michaels and Amasino
(2000) concluded that DNA replication, rather than cell division, Is required for
vemalization to occur.

Juvenility is the early developmental phase reported in various species
during which plants are unable to respond to favorable exogenous factors
promoting ﬂowering such as photoperiod and vemalization (Thomas and Vince-
Prue, 1984). Hence, overcoming juvenility is a prerequisite for perceiving
vemalization treatment in such species. In species such as Hedera helix L.
(English ivy) the juvenile and adult phase is easily distinguishable due to
differences in leaf morphology and arrangement (Amasino, 2002). However, in
many herbaceous perennial species such as Coreopsis grandiﬂora Hogg ex
Sweet 'Sunray' (largeﬂower tickseed), the number of leaves or nodes unfolded is
typically used to determine the end of juvenile period due to the lack of visual
indicators. Juvenility of Coreopsis grandiﬂora 'Sunray' seedlings apparently ends
with the formation of 8 nodes (16 leaves) and, subsequently, plants perceive
vemalization treatment (Yuan et al., 1998).

Localization of Vernalization. Lang (1965) reviewed following localized
cooling and grafting experiments performed to determine the primary site of
perception of vemalization. When various plant parts were cooled locally,
vemalization occurred when buds, embryos and excised meristems were
speciﬁcally cooled. Additionally, grafting various parts of vernalized plants onto
non-vernalized plants revealed that ﬂowering occurred only when vernalized

apical tissue was grafted on an non-vernalized plant. Additionally, excision of

12

vernalized apical tissue and replacing it with apical tissue from a non-vernalized
plant did not promote ﬂowering. Taken together, these results suggest that shoot
apical tissue, presumably the meristem, was the primary site of perception of
thermoinductive temperatures during vemalization. However, in these studies, it
was impossible to differentiate if the vemalization response occurred at the shoot
apical meristem (SAM), young leaves, or both (Searle et al., 2006). Determining
the site of vemalization was further complicated by reports that young leaves of
Lunan‘a annua (Wellensiek 1964) and mature leaves of Thlaspi arvense could be
vernalized (Metzger, 1988). Consequently, depending on the species, the
localization of vemalization is in the SAM and/or leaves. Based on this
conclusion, the regulation of vemalization may involve the ability of SAM to
respond to ﬂoral stimulus and/or the ability of leaves to generate a ﬂoral stimulus
(McDaniteI, 1985 cited by Searle et al., 2006).

Thermosensory Pathway. Exposure to low temperatures results in many
biochemical changes including changes in plasma membrane ﬂuidity,
modiﬁcation of membrane lipid composition, rearrangement of microﬁlaments,
Ca2+ ﬂuxes and cascades of protein phosphorylation (reviewed by Sung and
Amasino, 2005). Evidence indicates that Ca2+ ﬂuxes and cascades of protein
phosphorylation play a role in activation of cold acclimation response in plants
(Thomashow, 2001). As discussed earlier, cold acclimation and vemalization
pathways are distinct in Arabidopsis. The only possible molecular link between
these two pathways is HIGH EXPRESSION OF OSMO TICALLY RESPONSIVE

GENES1 (HOS1), which seems to be the upstream repressor of genes involved

13

in cold acclimation and vemalization pathway (Lee et al., 2001). However, the
components and mechanism of thermosensory pathway of Arabidopsis are yet to
be determined.

Time-keeping Mechanism. In addition to temperature sensing,
vemalization also involves a time-keeping mechanism that measures the
duration of exposure to thermoinductive temperatures during vemalization.
Based on their functions, the thermosensory pathway and time-keeping
mechanism probably share some common features. Although the molecular
components involved in this time-keeping mechanism are still unknown, recent
investigation of quantitative regulation of Arabidopsis vemalization genes have

provided some insight about this mechanism that will be discussed later.

Effective Therrnoinductive Temperatures

The efﬁcacy of vemalization treatment depends on temperatures and
insufﬁcient vemalization can result in incomplete ﬂowering. The thermoinductive
temperatures can be described as cardinal temperatures for vemalization with
minimum (Tmin), maximum (T max) and optimum (Tom) values (Atherton et al., 1990;
Yan and Hunt, 1999). Tmin and Tmax describe the upper and lower thresholds at
which the rate of progress to vemalization is zero (Atherton et al., 1990). Tmin
and Tmax are species-speciﬁc and range between >-4 °C and <17 °C,
respectively (Lang, 1965). Top, describes the range of optimum thermoinductive
temperatures at which maximum vemalization response is achieved (Atherton et

al., 1990). Salisbury and Ross (1992) reported that the Top. of Secale cereale L.

14

'Petkus' (winter rye) is about 0 to 10 °C following 6-week vemalization treatment,
however it appears that based on highest relative ﬂowering, Top, of Secale

cereale 'Petkus' is between 0 to 8 °C (Fig. 2).

 

 

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IIIIIIIIIIILILIIIIIIII

 

 

 

0
~6-4-2 0 2 4 6 810121416
temperature (°C) during vemalization treatment

Figure 2. Tom of Secale cereale 'Petkus' as a function of vemalization
temperature provided to imbibed seeds for 6 weeks (from Salisbury and Ross,
1992)

Similar to Tmin and Tmax, Tom is also species-speciﬁc. For example, the Top,
resulting in the greatest ﬂowering response of Veronica spicata L. 'Red Fox'
(spiked speedwell) and Isotoma axillan's Lindl. (blue star) were -2.5 °C and 5 to
10 °C, respectively (Fausey, 2005). Tom is relative and even within a species, it

depends on the duration of the vemalization treatment. For example, Veronica

spicata 'Red Fox' ﬂowered completely following 4 weeks at -2.5 to 0 °C however,

15

after 6 weeks, the effective thermoinductive temperature range broadened to
-2.5 to 2.5 °C, and further to -2.5 to 7.5 °C after 8 weeks (Fausey, 2005).
Additionally, by deﬁnition, Top, is also based on the vemalization response being
assessed (Rawson et al., 1998). For example, when vemalization response was
assessed as minimum days from sowing to heading, the Top, for Triticum
aestivum L. 'Dollarbird' (common wheat) was 19 °C. However, when
vemalization response was assessed as the minimum thermal time from sowing
to heading above the base temperature of 0 °C, Top, of the same wheat cultivar
was 6 °C (Rawson et al., 1998).

During vemalization, the exposure to thermoinductive temperatures and
observed ﬂowering response are temporally separated and hence, there is no
other indicator of completion of the vemalization process besides the subsequent
ﬂowering. Since insufﬁcient vemalization clue to the use of non-inductive
temperatures or short durations can elicit incomplete ﬂowering, empirical studies
investigating the effective vemalization temperatures and durations are essential
to ensure successful ﬂowering. Top, for various ﬂowering responses has been
empirically determined in ﬁeld crops including Triticum aestivum, Daucus carota
L. (carrot) and Allium cepa L. (onion; Brooking IR, 1996; Robertson et al., 1996;
Yan and Hunt, 1999; Streck, 2003) and select potted ﬂowering plants including
Cineran'a sp. and Lilium Iongiﬂorum Thunb. (Easter lily; Lange and Heins, 1990;

Yeh et al., 1997a; Streck and Schuh, 2005).

16

Quantitative Nature of Vernalization Response

As discussed earlier, the duration of exposure to thermoinductive
temperatures affects the intensity of the ﬂowering response and increasing the
duration of vemalization treatment increases the ﬂowering response until
saturation (Lang, 1965). This quantitative nature of vemalization has been
documented in plants exhibiting obligate and facultative vernalization responses
and has been demonstrated by investigating various ﬂowering parameters
including percentage of ﬂowering plants in a population, node or leaf
development until ﬂowering, time to ﬂower (Fig. 3), number of ﬂowers and

reproductive laterals (Fausey, 2005; Lang,1965; Lange and Heins, 1990).

140 H
120 ~

100 -

Time to anthesis (d)
I

60 F

 

40

l l l l l

0 10 20 30 40 50
Duration of cold (d)

Figure 3. The quantitative vemalization response of Petkus winter rye assessed
by measuring time to ﬂower computed after the end of thermoinductive
treatments (adapted from Vince-Prue, 1975).

17

Typically, at a constant thermoinductive temperature, the optimum
duration of vemalization treatment changes based on the response being
assessed. For example, 4-week treatment at -2.5 °C was sufﬁcient for achieving
100% ﬂowering of Veronica spicata 'Red Fox' however, longer durations at -2.5
°C increased the number of reproductive laterals, improving the ornamental value
at ﬂowering. Therefore, the choice of appropriate duration of vemalization

treatment should be selected to maximize all ﬂowering responses of signiﬁcance.

Memory of Vernalization

Once achieved, the vernalized state is maintained for a prolonged period
of time until conditions favorable for ﬂowering arise (Lang, 1965). Thus, a
cellular “memory” of the winter or exposure to prolonged cold is maintained
through several mitotic divisions until environmental conditions are favorable.
Meiosis resets the vemalization requirement in annuals and biennials, requiring
progeny of vernalized plants to go through vemalization again to attain the
capacity to ﬂower (Michaels and Amasino, 2000). Thus, the vernalized state is
epigenetically regulated. Perennials require vemalization to ﬂower in the next
season without going through meiosis, hence probably have a distinct
vemalization resetting mechanism. Vince-Prue (1975) proposed that the
vemalization requirement may be reset in perennials by devernalization
(discussed below) of some buds during summer, by the inability of some buds to
vernalize during vemalization treatment, by vemalization not being an

autocatalytic process, or by a combination of all these processes.

18

Devernalization

The complete or partial reversion of vemalization is termed as
devernalization and exposure to high temperatures following vemalization has a
devernalizing effect that increases with the temperature and duration of the
exposure (Lang, 1965). Devernalization has been documented in many crops
including Lactuca sem'ola (wild lettuce; Marks and Prince, 1979), Daucus carota
(Hiller and Kelly, 1979), Brassica oleracea var. botrytis (cauliﬂower; F ujime and
Hirose, 1980), Brassica oleracea var. gongylodes (kohlrabi; Wiebe et al., 1992),
Apium graveolens var. dulce (celery; Booi and Meurs, 1993), Allium ﬁstulosum
(Japanese bunching onion; Yamasaki et al., 2000) and Cineran'a sp., a potted
ﬂowering crop (Yeh et al., 1997b). The ranges of vernalizing, neutral (having
neither vernalizing nor devernalizing effect) and devernalizing temperatures can
be fairly close to one another and are species-speciﬁc (Lang, 1965). Attaining
saturation of vemalization typically renders the high temperature devernalization
ineffective (Lang, 1965). Additionally, exposure to neutral temperatures after
non-saturating vemalization treatment and prior to high temperature exposure
prevents devernalization. This effect is known as stabilization of vemalization
and has been demonstrated in Hyoscyamus niger L. (henbane), Secale cereale
'Petkus' and Arabidopsis (reviewed by Lang, 1965). In some species certain
conditions are either essential for devernalization or to maximize the
devernalizing effect including particular stage of development, preventing seed
germination by water supply restriction and low light intensity or absence of light

during high temperature exposure (Lang, 1965). For example, Lactuca sem’ola

19

plants can not be devernalized but seeds can be devernalized by exposure to 25

°C only prior to germination and in the absence of light (Marks and Prince, 1979).

Substitution of Vernalization Requirement

In some species, the vemalization requirement can be completely or
partially substituted by exposure to SD or LD photoperiods. SD can substitute for
vemalization requirement in some LD plants, hence such species can be
considered as short-long-day plants or vemalization requiring LD plants (Vince-
Prue, 1975). Based on experiments investigating the effect of inductive
treatments of SD or vemalization prior to exposure to LD in Campanula medium
and Silene annen'a L. (sweet-William catchﬂy), it was postulated that these
inductive treatments operated through different pathways and resulted in the
same outcome (reviewed by Vince-Prue, 1975). However, Dubcovsky et al.
(2006) reported that in winter wheat, which ﬂowers in response to SD or
vemalization followed by LD, exposure to SD downregulated vemalization
responsive ﬂowering inhibitor, VRN2 and ﬂowering promoter VRN1 was
upregulated only after exposure to LD. Therefore, the mechanism of substitution
of vemalization by SD involves regulation of vemalization responsive genes.

Ketellapper and Barbaro (1966) reported that Coreopsis grandiﬂora 'Single
Mayﬁeld Giant' could be induced to ﬂower either by vemalization or SD treatment
followed by LD. In this study, vemalization was more effective than SD in
inducing more plants in the population to ﬂower and ﬂowering was faster after

vemalization than SD treatment. Damann and Lyons (1993) reported that

20

Coreopsis grandiﬂora 'Sunray' did not ﬂower in response to 10-week SD
treatment (9-h photoperiod) followed by LD however, Runkle et al. (unpublished
data) found that 100% Coreopsis grandiﬂora 'Sunray' ﬂowered following the
same treatment. Exposure to LD can partially substitute for vemalization
requirement in Easter lily. In the ﬁeld, Easter lily bulbs receive some
vemalization prior to harvest and exposure to LD can substitute for partial
vemalization (Rees, 1987). High daily light integral also partially substituted for
vemalization in Salvia xsuperba Stapf 'Blaukonigin' (perennial sage; Waaseth et

aL,2006)

Vernalization in Arabidopsis

Arabidopsis thaliana has been used as a model system to study ﬂowering
and in recent years, signiﬁcant progress has been made in understanding the
genetic and molecular basis of ﬂowering in Arabidopsis. In Arabidopsis, four
pathways involved in transition of the vegetative SAM to reproductive meristem
have been identiﬁed. These pathways include vemalization pathway,
autonomous or constitutive pathway, photoperiod pathway and GA-dependant
pathway (Simpson et al, 1999) (Fig. 4). Flowering of Arabidopsis is promoted by
exposure to vemalization and LD in a facultative manner (Napp-Zinn, 1969). The
vemalization pathway and photoperiod pathway integrate the external stimuli to
coordinate ﬂowering with favorable environmental conditions. In contrast, the
autonomous pathway and GA-dependant pathway act independent of the

environmental conditions. There is some redundancy and cross-talk between all

21

the ﬂowering pathways ensuring success in ﬂowering under diverse

environmental and developmental conditions (Amasino, 1996).

Vegetative

Development
Photoperiod Autonomous

pathway \ / pathway
GA-dependent/ \

Short-day _ Vernalization
pathway Flowering

Figure 4. Pathways regulating transition from vegetative development to
ﬂowering in Arabidopsis thaliana (from Michaels and Amasino, 2000).

As mentioned earlier, Arabidopsis has a wide geographic distribution
resulting in natural variation in ﬂowering time. Based on the natural variation in
vemalization requirements, Arabidopsis ecotypes can be grouped into two
categories: early ﬂowering ecotypes that behave as summer annuals and late
ﬂowering ecotypes that behave as winter annuals. Vernalization has no effect on
ﬂowering of the early ﬂowering ecotypes whereas, vemalization accelerates
ﬂowering of the late ﬂowering ecotypes (Fig. 5). This natural diversity in the
vemalization responsiveness of Arabidopsis ecotypes has been exploited in
many studies to understand the genetic and molecular basis of the vemalization
response. Studying Arabidopsis mutants with altered vemalization response has
been another common approach used to identify key regulatory genes involved
in vemalization. Extensive studies with early and late ﬂowering ecotypes and

vemalization mutants have resulted in identiﬁcation and characterization of

22

FRIGIDA (FRI) and FLOWERING LOCUS C (FLC), two key genes involved in

Arabidopsis vemalization pathway.

(3) (b) (6)

Winter annual Winter annual

Summer annual , . .
w/o vemalization w/ vemalization

 

Figure 5. Natural variability in ﬂowering time of Arabidopsis thaliana ecotypes.
(a) Summer annual types are naturally early ﬂowering and do not respond to
vemalization treatment, (b) ﬂowering of winter annual types is delayed without
vemalization treatment, (0) winter annual types ﬂower rapidly similar to summer
annuals following vemalization treatment (from Sung and Amasino, 2004a).
FRIGIDA (FRI). Napp-Zinn (1955) ﬁrst described the FRIGIDA (FRI)
locus responsible for delayed ﬂowering in late-ﬂowering Arabidopsis ecotypes by
analyzing the progeny of early-ﬂowering and late-ﬂowering ecotypes (cited by
Johanson, 2000) and further studies showed that FR/ is a dominant gene (Napp-
Zinn, 1987). Cloning of FRI revealed that a single copy of FRI is present in
Arabidopsis and the predicted FRI protein is not homologous to any known
protein (Johanson, 2000). Johanson et al. (2000) identiﬁed three lesions in FRI
by studying 40 Arabidopsis ecotypes with varying ﬂowering responses and the
presence of at least one FRI lesion was the primary basis for the spring annual

ﬂowering response in the evaluated ecotypes. The study by Johanson et al.

(2000) also showed that the lesions in FRI occurred as at least two independent

23

evolutionary events, indicating a strong selection against vemalization in some
environments.

FLOWERING LOCUS C (FLC). Segregation analysis revealed the
requirement of a dominant allele on chromosome 5 for late ﬂowering phenotype
of dominant FRI and this locus was named FLOWERING LOCUS C (FLC)
(Koornneef et al., 1994). FLC expression has been detected in leaves, apical
tissue and roots of Arabidopsis (Michaels and Amasino, 1999). FLC is a
dominant MADS box transcription factor responsible for repression of ﬂowering in
the absence of vemalization in late ﬂowering Arabidopsis ecotypes (Michaels and
Amasino, 1999; Sheldon et al., 1999). FLC is upregulated in vemalization-
responsive mutants and FLC levels mostly remain unchanged in vernalization-
nonresponsive mutants (Sheldon et al., 1999). FRI increases FLC mRNA levels
in late ﬂowering Arabidopsis lines and following vemalization, FLC mRNA levels
are undetectable. This downregulation of FLC correlates with elimination of the
late ﬂowering phenotype (Michaels and Amasino, 1999). FLC acts downstream
of FRI (Michaels and Amasino, 2001) and vemalization eliminates the FRI-
mediated elevation of FLC expression, resulting in early ﬂowering phenotype.
FRI and FLC regulate the vemalization response in Arabidopsis by acting
synergistically, delaying ﬂowering in late ﬂowering ecotypes in the absence of
vemalization and a loss of function mutation in FRI or FLC results in early
ﬂowering (Michaels and Amasino, 1999).

Dose-dependant response of FLC. In non-vernalized ecotypes or

mutants of Arabidopsis, vemalization response depends on the levels of FLC

24

transcripts and the duration of cold required to saturate vemalization response is
proportional to the extent of downregulation of FLC transcripts (Sheldon et al.,
2000b). Michaels and Amasino (2000) reported that by adding additional copies
of FLC, late ﬂowering Arabidopsis containing FRI can be converted into a true
biennial that does not ﬂower without a vemalization treatment. Thus, FLC acts in
a dose-dependent (rheostat-like) manner to delay ﬂowering in the absence of
vemalization (Fig. 6) (Michaels and Amasino, 2000). Therefore, differences in
molecular mechanism may not be required for the regulation of obligate versus
facultative vemalization response, only different levels of ﬂowering repressor can

be adequate (Sung and Amasino, 2005).

A. air-

ﬂc/FLC FLC/FLC

n9 FLC *
tic/tic f IGP%

Additional copies
of FLC

 

Annual Biennial

FLOWERING RH EOS'I'AT

Figure 6. Dose-dependent (rheostat-like) response of FLC in delaying ﬂowering
in Arabidopsis thaliana. In FRI background, ﬂowering is delayed based on the
number of FLC copies and transgenic plants with additional FLC copies behave

as biennials in absence of vemalization treatment (from Michaels and Amasino,
2000).

25

Role of FLC in Quantitative Nature of Vernalization Response. As
discussed earlier, vemalization is a quantitative process and with increasing
durations at thermoinductive temperatures, vemalization response increases until
saturation. In Arabidopsis, there exists a quantitative relationship between the
duration of vemalization treatment, ﬂowering time and FLC transcript levels,
indicating that FLC plays a key role in quantitative vemalization response
(Sheldon et al., 2000b). Until recently, it was unclear whether the degree of FLC
downregulation resulting in quantitative vemalization response was due to the
extent of initial FLC repression or the degree of maintenance of FLC repression
during the subsequent plant development. Recently, Sheldon et al. (2006)
reported that following vemalization treatment, the extent of initial repression of
FLC depends on the duration of exposure to thermoinductive temperatures.

Genes Regulating FLC Repression. Mutagenesis identiﬁed early
ﬂowering and vemalization insensitive mutants vemalization1 (vm1) and
vemalization2 (vm2) (Chandler, 1996). VRN1 encodes a DNA-binding protein
with 83 domains (Levy et al., 2002) and VRN2 encodes a zinc ﬁnger protein
homologous to Drosophila polycomb group (PcG) protein, Su(Z)12 (Gendall et al.,
2001). Since in PcG mutants of Drosophila, gene repression occurs but fails to
be maintained, it was proposed that VRN2 played a similar role in Arabidopsis
and was not responsible for repressing FLC during vemalization treatment but
was involved in maintaining the repressed state of FLC after the plants were
returned to warmer temperatures (Gendall et al., 2001 ). Levy et al. (2002)

suggested that similar to VRN2, the role of VRN1 was to maintain the repressed

26

state of FLC. However, recently Sheldon et al. (2006) showed that both VRN1
and VRN2 activities are needed for maximal repression of FLC during
vemalization and inactive VRN1 or VRN2 reduce vemalization response, with
VRN2 having a greater effect than VRN1. Sheldon et al. (2006) also showed that
VRN1 and VRN2 are not required to maintain the repressed state of FLC
although the authors suggested the possibility that VRN1 and VRN2 may
maintain FLC repression initiated by other factors in wild type plants.

Mutagenesis also identiﬁed vemalization independent 3 (vin3) in which
vemalization response is completely blocked. VIN3 gene has been cloned and
encodes a PHD (plant homeodomain) ﬁnger containing protein, which is known
to be involved in chromatin remodeling (Sung and Amasino, 2004b). VIN3 can
distinguish between transient exposure to cold and prolonged cold exposure and
is expressed only after prolonged exposure to cold (Sung and Amasino, 2004b)
Therefore, it is postulated that VIN3 plays a key role in affecting ﬂowering time
after the end of the winter (Sung and Amasino, 2004a). Since VIN3 is expressed
only after exposure to cold for a duration needed to satisfy vemalization
requirement, it can serve as a marker of vemalization duration essential for
successful ﬂowering.

Epigenetic Regulation of FLC. As discussed earlier, vemalization is
epigenetically regulated and its "cellular memory" persists through mitotic
divisions and is reset Upon meiosis. In Arabidopsis, after vemalization FLC is
downregulated and its levels remain unchanged through mitosis. The FLC levels

are reinstated after meiosis, reforming the vemalization requirement. Therefore,

27

FLC acts as an epigenetic switch regulating vemalization response (Michaels
and Amasino, 1999). Vernalization causes sequence-speciﬁc changes in the
histone modiﬁcation of FLC and these changes result in repression of FLC
(Bastow et al., 2004). The changes in FLC histone modiﬁcation include
deacetylation of lysine 9 (K9) and lysine 14 (K14) on histone H3 (Sung and
Amasino, 2004b), followed by dimethylation of lysine 27 (K27) and K9 on histone
H3 (Bastow, 2004; Sung and Amasino, 2004b). The discovery of VRN1, VRN2
and VIN3 have provided insight into the mechanism of histone modiﬁcations of
FLC by these three genes, leading to proposed models of epigenetic regulation
of FLC.

Based on the initially proposed role of VRN1 and VRN2 in maintenance of
FLC repression, Sung and Amasino (2004a; 2005) suggested a model of
epigenetic regulation of vemalization in Arabidopsis. According to this model,
during prolonged exposure to low temperatures, VIN3 initiates FLC histone
deacetylation, and hence the initial FLC repression. This histone deacetylation
generates an environment favorable for methylation of K27 and K9 on histone H3
involving VRN1-IVRN2-containing complex. It has been proposed that
methylation of Histone H3 at K9 promotes stable heterochromatin formation by
HETEROCHROMA TIN PROTEIN1 (HP1) in animals. Since the repression of
FLC following vemalization is unstable in Arabidopsis hp1 mutants, HP1 may be
involved in maintaining FLC repression following vemalization. Thus, the
epigenetic regulation of vemalization involves a cascade of histone modiﬁcations

of FLC following vemalization, resulting in mitotically stable repression of FLC

28

heterochromatin serving as the "cellular memory" of the winter. Recently,
Sheldon et al. (2006) showed that methylation of K27 and K9 on H3 may not be
required for maintaining FLC repression after the end of vemalization treatment
and postulated that other proteins may be involved in maintaining FLC repression.
Further studies are necessary to determine the components involved in the
mechanism of epigenetic regulation of FLC.

Downstream Target Genes of FLC. In vemalization-responsive
ecotypes of Arabidopsis, ﬂowering is delayed by FLC-mediated repression of
ﬂoral integrator genes SUPPRESSOR OF OVEREXPRESSION OF
CONSTANS1 (SOC1)/ AGAMOUS LIKE20 (AGL20) (Lee et al., 2000) and
FLOWERING LOCUS T (FT) (Michaels et al., 2005). In their proposed ﬂowering
regulation model, Michaels et al. (2005) noted that FLC-mediated repression of
SOC1 is stronger than that of FT. SOC1 and FT promote ﬂowering by activating
ﬂoral meristem identity genes such as LEAFY (LFY) and APE TALA (AP1)
(reviewed by Sung and Amasino, 2005). Additionally, FT also regulates ﬂoral
meristem identity genes by activating SOC1 (Michaels et al., 2005). A schematic
diagram showing the primary genes involved in FLC-mediated vemalization

pathway in Arabidopsis is presented in Fig. 7.

29

Vernalization

I

VFIN 1, VFN2, VIN3 etc

I

FLC

I

8001, FT etc

I

LFY AP1 etc

I

Vegetative —> Flowering

Figure 7. Vernalization pathway regulated by FLC in Arabidopsis thaliana.
Vernalization induces VRN1, VRN2 and VIN3 genes which are repressors of FLC.
FLC in turn is a repressor of SOC1 and FT which are promoters of meristem
identity genes such as LFY and AP1 (from Sung and Amasino, 2005).

FLC Regulation by Autonomous Pathway Genes. The autonomous
pathway consists of FCA, FY, FPA, F VE, LUMINIDEPENDENS (LD) and
FLOWERING LOCUS D (FLD) genes. Mutants of autonomous pathway genes
are delayed in ﬂowering irrespective of the photoperiod (Koornneef et al., 1991)
and ﬂowering of these mutants is hastened by vemalization treatment (Lee and
Amasino, 1995). The delayed ﬂowering of autonomous pathway mutants
suggested that the autonomous pathway genes may play a role of in promotion

of ﬂowering. Furthermore, the hastening of ﬂowering of autonomous pathway

mutants following vemalization treatment indicated that these ﬂowering promoter

30

genes acted in a pathway parallel to the vemalization pathway (reviewed by
Henderson et al., 2003). Higher levels of FLC RNA in autonomous pathway
mutants suggested that these genes repress FLC and this role was substantiated
by complete suppression of the late ﬂowering phenotype in double mutants of ﬂc
and autonomous pathway genes (reviewed by Henderson et al., 2003). Recently
it was shown that autonomous pathway consists of a group of genes involved in
a non-linear pathway regulating the expression of FLC (reviewed by Sung and
Amasino, 2005).

Other Genes Regulating Arabidopsis Vernalization Response.
Signiﬁcant progress has been made in understanding the molecular regulation of
FLC-mediated vemalization pathway in Arabidopsis. However, the role of
additional regulators of Arabidopsis vemalization pathway remains to be
elucidated. Some of these additional regulatory genes in Arabidopsis
vemalization pathway are brieﬂy described below.

Although FRI is the key gene involved in upregulating FLC in late
flowering Arabidopsis ecotypes, additional regulators of FLC may exist. For
example, in Sy-O accession of Arabidopsis, AERIAL ROSE TTE 1 (ART1)
upregulated FLC by a mechanism distinct from that of FRI and ART1 and FLC
synergistically repressed ﬂowering (Poduska, 2003). Although both FRI and
ART1 upregulate FLC, the similarity of their mechanism is unknown (reviewed by
Henderson et al., 2003).

While the role of FLC as a key regulatory gene of vemalization pathway in

Arabidopsis remains unchallenged, evidence of an FLC-independent

31

vemalization pathway also exists. Michaels and Amasino (2001) reported that
when ﬂc null mutants were grown under SD, their ﬂowering was promoted by
vemalization, indicating the existence of an FLC-independent pathway. This
promotion of ﬂowering under SD was completely blocked in vin3 ﬂc null double
mutant, suggesting that VIN3 is involved in FLC-dependent and FLC-
independent vemalization pathways (Sung and Amasino, 2004b). The molecular
mechanisms involved in this FLC-independent vemalization pathway remains to
be elucidated.

It was previously discussed that VIN3 plays a key role in FLC repression.
However, overexpression of VIN3 does not abolish the vemalization requirement
for promotion of ﬂowering and hence, the process of FLC repression may involve
additional regulatory factors (Sung and Amasino, 2004b). Also, the regulatory
factors involved in the induction of VIN3 in response to prolonged exposure to

thermoinductive temperatures still remain unknown.

Vernalization in Wheat

The vemalization pathway in wheat has been shown to be distinct from
the FLC-mediated vemalization pathway in Arabidopsis. In both Triticum
monococcum L. (diploid wheat) and Triticum aestivum (tetraploid wheat), VRN1
and VRN2 are the two key regulatory genes governing the vemalization
response. Note that VRN1 and VRN2 in wheat are distinct from VRN1 and
VRN2 in Arabidopsis. Dominant alleles of VRN1 are required for spring annual

growth habit whereas, dominant alleles of VRN2 are required for winter annual

32

growth habit of wheat (Yan et al., 2003; Yan et al., 2004). In the absence of
vemalization treatment, VRN2 delays ﬂowering by repressing VRN1 and
vemalization treatment promotes ﬂowering by downregulating VRN2, thereby
relieving the block on VRN1 (Yan et al., 2003; Yan et al., 2004) (Fig. 8). Thus,
VRN2 in wheat seems to play a role similar to that of FLC in Arabidopsis.
Presence of dominant alleles of VRN1 and VRN2 results in spring annual growth
habit in wheat as dominant alleles of VRN1 can not be repressed by VRN2 (Yan
et al., 2003). Orthologues of wheat vemalization genes have been reported in
Hordeum vulgare L. (barley; Dubcovsky et al., 1998) and therefore, it has been
postulated that the vemalization pathway mediated by VRN1 and VRN2 genes is
conserved in monocots.

Recessive alleles of Flowering without Spring annual

VRN1 and VRN2 Vernalization treatment growth habit
Recessive allele of VRN1 Flowering only after ......... Winter annual
and dominant allele of VRN2 Vernalization treatment growth habit

VRN2I— Vernalization

I

VRN1
Dominant alleles of Flowering without ......... Spring annual
VRN1 and VRN2 Vernalization treatment growth habit

Figure 8. Vernalization pathway mediated by VRN1 and VRN2 is responsible for
spring annual and winter annual growth habit in wheat.

33

Future Perspectives

The molecular mechanism of vemalization in Arabidopsis involving FLC
regulation has been shown to extend to some other species belonging to the
Brassicaceae family including Brassica juncea (L.) Czern. (Indian mustard;
Martynov and Khavkin, 2004), Brassica napus L. (rape; Tadege et al., 2001),
Brassica oleracea L. var capitata (cabbage; Lin et al., 2005), Brassica rapa
(turnip; Schranz et al., 2002) and Thellungiella halophila O. E. Schulz (salt cress;
Fang et al., 2006). In these species, homologues or orthologues of FLC have
been found and are believed to mediate the vemalization response. Thus, so far
the FLC-mediated vemalization pathway is limited to the members of Brassica
family and has not been extended to the plethora of vemalization-responsive
species from other families. As previously mentioned, the vemalization pathway
in monocots including wheat and barley is distinct from the vemalization pathway
in dicots, essentially represented by select Brassicaceae species. Studies
investigating the molecular mechanism of vemalization in a winder range of

species could account for the molecular basis of vemalization response in plants.

34

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42

CHAPTER II:
THE FLOWERING RESPONSE BEING ASSESSED REGULATES THE
OPTIMUM TEMPERATURE FOR VERNALIZING CAMPANULA ‘BIRCH

HYBRID’

43

The Flowering Response Being Assessed Regulates the Optimum

Temperature for Vernalizing Campanula ‘Birch Hybrid’

Sonali Padhye and Arthur Cameron

Department of Horticulture, Michigan State University, East Lansing, MI 48824

Additional index words. cardinal temperatures for vemalization, herbaceous

perennial, thermal time to ﬂower

Abstract

The objective of this study was to characterize the effect of vemalization
temperature and duration on various ﬂowering responses of Campanula ‘Birch
Hybrid’. Clonally propagated plants of Campanula 'Birch Hybrid’ were exposed
to -2.5, 0, 2.5, 5, 7.5, 10, 12.5, 15, 17.5 or 20 °C for 0, 3, 5, 7, 9 or 12 weeks and
subsequently grown at 20 °C in a greenhouse. Campanula 'Birch Hybrid'
exhibited a near-obligate vemalization requirement and all ﬂowering responses
studied were affected by the vemalization temperature, duration and their
interaction. The minimal and maximal cardinal temperatures for vemalization
were <0 °C and between 15 and 17.5 °C, respectively. At each treatment
duration, the range of optimal temperatures (Tom) varied based on the ﬂowering
response assessed and ranged between 0 to 12.5 °C for ﬂowering percentage to

5 to 7.5 °C for rate of progress to ﬂowering. Additionally, Tom for ﬂowering time

44

also varied when analyzed as rate of progress to ﬂowering, time to ﬂower from
the end of temperature treatments, total time to ﬂower measured from the start of
temperature treatments and thermal time to ﬂower measured from the initiation of
temperature treatments as average daily temperature above the base
temperature. For example, following 12-week treatment, Top. for thermal time to
ﬂower was 7.5 °C and for time to ﬂower and total time to ﬂower the range of Top:
broadened to 2.5 to 12.5 °C. Since the ﬂowering response assessed altered the
Tom, this study reiterates the signiﬁcance of considering all relevant ﬂowering

responses while developing and interpreting vemalization models.

Introduction

Vernalization is deﬁned as the promotion of ﬂowering following cold
treatment (Chouard, 1960; Vince-Prue, 1975; Thomas and Vince-Prue, 1984).
The intensity of vemalization response is a function of the interaction between
thermoinductive temperature and duration of exposure. The thermoinductive
temperatures vary by species (Lang, 1965) and have been described as cardinal
temperatures for vemalization with minimum (Tmin), optimum (Tom) and maximum
(Tmax) values (Atherton et al., 1990; Yan and Hunt, 1999). Tm,n and Tmax are
minimum and maximum temperatures at which the rate of vemalization is zero.
The range of vernalizing temperature deﬁned by Tm," and Tmax is species-
dependant and quite broad (-4 to 17 °C) (Table 1). Top, is the range of

vernalizing temperatures at which highest vemalization response is achieved and

45

is also species-speciﬁc (Table 1). By deﬁnition, Tom is relative and is based on
the vemalization response being assessed.

The cardinal temperatures for vemalization are also relative due to their
dependence on treatment duration (Lang, 1965). Duration of thermoinductive
treatment has a quantitative effect on vemalization response until saturation and
therefore, increasing durations of thermoinductive treatment progressively
increase ﬂowering response until the response saturates (Lang, 1965). Optimum
temperature and duration for vemalization have been characterized for several
ﬁeld crops including Triticum aestivum L., Daucus carota L. and Allium cepa L.
(Brooking, 1996; Robertson et al., 1996; Streck, 2003; Yan and Hunt, 1999) and
select potted ﬂowering plants including Cineran'a sp. and Lilium Iongiflorum
Thunb. (Lange and Heins, 1990; Streck and Schuh, 2005; Yeh et al., 1997).
Flowering responses studied include the number of plants ﬂowering in a
population (Clough et al., 2001; Lange and Heins, 1990), node or leaf number at
ﬂowering (Brooking IR, 1996; Clough et al., 2001; Lange and Heins, 1990;
Rawson et al., 1998; Robertson et al., 1996; Suzuki and Metzger, 2001), rate of
ﬂowering (Streck, 2003; Yan and Hunt, 1999), time to ﬂower (Clough et al., 2001;
Rawson et al., 1998; Streck et al., 2003; Suzuki and Metzger, 2001; Yan and
Hunt, 1999), thermal time to ﬂower (Rawson et al., 1998), number of buds and
ﬂowers at ﬂowering (Clough et al., 2001; Suzuki and Metzger, 2001) and percent
reproductive lateral nodes (Fausey, 2005). Although many studies have

determined Top, based on thermoinductive temperature, treatment duration and

46

one or more ﬂowering responses listed above, to our knowledge, no study has
yet characterized Tom for all the listed ﬂowering responses in one species.

Campanula ‘Birch Hybrid’, a hybrid of Campanula portenschlagiana Schult.
and Campanula poscharskyana Degen, is an herbaceous perennial that has
been reported to exhibit a qualitative vemalization response (Enﬁeld, 2001; Niu
et al., 2004). Campanula ‘Birch Hybrid’ is a good candidate for investigating the
vemalization responses since it can be clonally propagated and produces large
numbers of propagules from a few stockplants. While stockplants are held in a
controlled environment, propagation and thermoinductive treatments can be
scheduled at varying times so that all treated plants can be transferred and
grown in a greenhouse simultaneously. Thus, all treated plants can be grown
under the same temperature and light conditions regardless of treatment duration
as demonstrated by Fausey (2005). Campanula ‘Birch Hybrid’ has a day-neutral
photoperiodic response and can be vernalized as small rooted cuttings. Niu et al.
(2004) characterized the vemalization response of Campanula ‘Birch Hybrid’ at 0
to 10 °C for 0 to 8 weeks based on ﬂowering percentage, time to visible bud and
ﬂower, number of ﬂowering shoots and number of ﬂower buds per shoot and
found that temperature of 0 to 10 °C was equally effective in promoting ﬂowering
following 6- or 8-week treatments. However, investigation of the responses of
Campanula 'Birch Hybrid' to a broader range of temperatures and durations is
lacking and the cardinal temperatures for a range of vemalization responses are
not established. The objective of this study was to characterize the effect of

various thermoinductive temperatures and durations on different ﬂowering

47

responses of Campanula “Birch Hybrid’. We also determined the Tm... and Tmax
for vemalization of Campanula ‘Birch Hybrid’ and characterized the Top, for a

range of ﬂowering responses.

Materials and Methods
Stockplant Management and Propagule Culture. Clonally propagated
Campanula ‘Birch Hybrid’ stockplants were grown for ~5 weeks in 13-cm square
plastic containers ﬁlled with commercial soil-less medium (Sure-Mix; Michigan
Grower Products, Galesburg, Mich.) and were rejuvenated by dividing and
repotting as necessary through the experiment. The stockplants were grown in a
controlled environment chamber set at 22 °C under a 13-h photoperiod provided
from 0600 to 1900 HR by incandescent (60A-13OV; Philips, Somerset, N.J.) and
ﬂuorescent lamps (VHOF96T12; Philips, Bloomﬁeld, N.J.) (~150 umol'm'z's'1
photosynthetic photon ﬂux; PPF). Stockplants were watered when necessary
with acidiﬁed well water (H2804 to a titratable alkalinity of ~140 mg'L'1 CaCOa)
containing nutrients (40 N, 4 P, 40 K, 5 Ca, 0.3 Fe, 0.2 Mn, 0.2 Cu, 0.03 B, 0.03
Mo, 0.2 Zn mgL"; MSU Special, Greencare Fertilizers, Chicago).

Shoot-tip cuttings with 4 to 6 nodes were taken starting 13 May 2003 and
17 September 2003 for replication 1 and 2 of the study, respectively. Additional
cuttings were taken 3, 5, 7, 9, and 12 weeks later during each replication. I
Cuttings were dipped in a commercial rooting hormone (Dip ‘N Grow; Clackamas,
Ore.) containing 1 g'L’1 indole-3-butyric acid and 0.5 g'L'1 naphthalene acetic

acid and rooted in 72-cell trays (50-mL cell volume; Landmark Plastic

48

Corporation, Akron, Ohio) containing 50% commercial peat-perlite media (Sure-
Mix; Michigan Growers Products, Galesburg, Mich.) and 50% coarse perlite
(Therrn-O-Rock; East Inc., New Eagle, Pa.). Cuttings were rooted for 20 d under
mist and weaned for 4 d by manual watering in a glass propagation house set at
23 °C air temperature and 26 °C media temperature, maintained by providing
bottom heat, and 0.3 kPa vapor pressure deﬁcit generated by injecting water
vapor in the air. A blackout system open from 0800 to 1700 HR maintained a 9-h
photoperiod in the propagation environment and the light was provided by natural
sunlight. Following propagation, plants were grown for an additional 33 d in a
controlled environment chamber maintained at 20 °C under the same lighting and

watering regimen as the stockplants.

Temperature Treatments. For each replication, 57 d from the start of rooting,
plants were transferred to controlled environment chambers set at -2.5, 0, 2.5, 5,
7.5, 10, 12.5, 15, 17.5 or 20 °C for 0, 3, 5, 7, 9 or 12 weeks. Incandescent and
ﬂuorescent lamps provided 20 (-2.5 to 2.5 °C) and 100 (5 to 20 °C) umol'm’z's”1
PPF for 11 h. During temperature treatments, plants were watered with acidiﬁed
well water with nutrients described above. Plants were transferred to the
temperature treatments on different dates for each treatment duration, such that
all of the treatments ended on the same day (1 October 2003 and 5 February

2004 for replications 1 and 2, respectively).

49

Plant Culture and Climate Control. Following temperature treatments, nine
plants per treatment combination and an additional nine plants as non-vernalized
controls were potted in 13-cm square plastic containers ﬁlled with commercial
soil-less medium (Sure-Mix) and grown in a glass greenhouse set at 20 °C under
a 16-h photoperiod. High-pressure sodium lamps provided supplemental lighting
(150 umol'm'z's’1 PPF) when ambient light was below 200 umol'm'z's"1 and
ceased after ambient light exceeded 400 umol'm'z's". When necessary, plants
were watered with reverse osmosis water containing nutrients (125 N, 12 P, 100
K, 65 Ca, 12 Mg, 1.0 Fe, 0.5 Mn, 1.0 Cu, 0.3 B, 0.1 Mo mg'L"; MSU Special,
Greencare Fertilizers, Chicago).

Greenhouse environmental control was regulated by a climate-control
computer (Priva, Model CD750; De Lier, The Netherlands). Air temperature was
measured at plant height on every greenhouse bench by type E thermocouples
(TT-E-40; Omega engineering, Stamford, Conn.) placed in aspirated tubes and
PPF at plant height was measured at two locations by line quantum sensors
containing 10 photodiodes (Apogee Instruments, Logan, Utah). Temperature
and light sensors were connected to a CR10 datalogger (Campbell Scientiﬁc,
Logan, Utah) and data were collected every 10 s and hourly averages were
calculated and recorded in a computer. The average daily temperature (ADT)
and daily light integral were calculated from the day of potting for 15 weeks and
were 20.8 and 21.0 °C and 9.0 and 13.2 mol‘m'z'd"1 during replication 1 and 2,

respectively.

50

Data Collection and Analysis. Number of nodes on each plant were counted
prior to and at the end of temperature treatments. Time to develop one node
during vemalization was computed and its reciprocal was used as the rate of
node development for regression analysis using PROC REG in SAS (SAS
Institute, Cary, NC). Data from the two replications were not signiﬁcantly
different and therefore were pooled in the analysis. The base temperature for
node development and the thermal time (°C d) required for developing one node
were computed as described by Roberts and Summerﬁeld (1987).

The date of ﬁrst open ﬂower was recorded and number of days from the
end of temperature treatments to ﬁrst open ﬂower were reported as time to ﬁrst
open ﬂower. Plants without an open ﬂower after 105 d in the greenhouse were
considered vegetative. Rate of progress to ﬂowering was computed by taking
the reciprocal of time to ﬁrst open ﬂower and the rate of progress to ﬂowering of
vegetative plants was reported as zero. Since vegetative plants were eliminated
from ﬂowering time analysis, even though the same dataset was used to
calculate rate of progress to ﬂowering and time to ﬂower, the analyses of these
two responses differed for the treatments that did not achieve 100% ﬂowering.
Total time to ﬂower was computed as days from the beginning of temperature
treatments to ﬁrst open ﬂower. The thermal time (°C d) to ﬂower of individual
ﬂowering plants was calculated as accumulation of ADT above the estimated
base temperature for leaf development from the beginning of temperature

treatments to the day of ﬁrst open ﬂower.

51

The number of vegetative and reproductive laterals was counted one
week after ﬁrst open ﬂower and percent reproductive laterals was computed.
One week after ﬁrst open ﬂower, the number of open ﬂowers were counted and
additionally during replication 1, for every 4’", 5th and 6th plant to ﬂower in each
treatment group, the number of ﬂower buds and ﬂowers were recorded. Due to
the time constraint imposed by counting the very large number of ﬂower buds
and ﬂowers present, number of ﬂower buds and ﬂowers were counted on 4‘", 5th
and 6th plants to ﬂower, in an attempt to measure the “average” potential of the
ﬂowering treatment group.

Days to ﬂower, rate of progress to ﬂowering, thermal time to ﬂower,
percent reproductive laterals, number of open ﬂowers and number of buds and
ﬂowers were analyzed using SAS’s (SAS Institute, Cary, NC) PROC MIXED
and the least signiﬁcant difference procedure was used for paired comparisons
with P=0.05 as a maximum value for signiﬁcance. The experiment was
completely randomized with temperature and duration as treatment factors in a
factorial arrangement. Pooled data from both replications were analyzed for all
response variables except number of open ﬂowers. Number of open ﬂowers
differed signiﬁcantly between the two replications and hence was analyzed

separately.
Results

Survival and Leaf Development During Vernalization. All plants treated at 20 °C

survived and showed no visual symptoms of injury during their subsequent

52

growth. Following exposure to -2.5 °C, 36% and 27% Campanula ‘Birch Hybrid’
plants died during replications 1 and 2, respectively, and several surviving plants
exhibited varying degrees of visual symptoms of freezing injury such as water-
soaked areas and necrosis. Therefore, all data from -2.5 °C treatments were
eliminated from analyses.

During temperature treatments, time to develop one node decreased in a
non-linear fashion with increasing temperature (Fig. 1A) and averaged 50.8 and
7.5 d at 0 and 20 °C, respectively. The rate of node development ﬁt a linear
regression model (r2=0.63, signiﬁcant at P=0.001) (Fig. 1B) and based on the
model, the base temperature and the thermal time (°C d) for node development

were estimated to be -2.9 °C and 151.5 °C d, respectively.

Flowering Response. No plants ﬂowered without temperature treatment (data
not shown) and only 1 out of 90 plants in each of the 17.5 and 20 °C treatments
ﬂowered following vemalization (Fig. 2A and B). Based on ﬂowering percentage,
Campanula ‘Birch Hybrid’ vernalized between 0 to 15 °C and the response was
quantitative with duration. For example, ﬂowering percentage at 15 °C increased
from 0 to 39% following treatment for 5 and 7 weeks, respectively and further
increased to 94% after 12 weeks. Complete (100%) ﬂowering of Campanula
‘Birch Hybrid’ was achieved following 5 weeks at 2.5 to 7.5 °C, 7 weeks at 0 to
7.5 and 12.5 °C, and 9 and 12 weeks at 0 to 12.5 °C. Incomplete ﬂowering

occurred after shorter durations at 0 to 15 °C.

53

Rate of Progress to Flowering, Time to Flower and Total Time to Flower. All
parameters relating to ﬂowering time were signiﬁcantly affected by treatment
temperature, duration and their interaction. The fastest rate of ﬂowering following
treatment for 5 to 9 weeks was achieved at 5 or 7.5 °C, and after 12-week
treatment at 7.5 °C (Fig. 3A). One plant each treated at 17.5 or 20 °C ﬂowered,
but ﬂowering was considerably delayed and the rate of progress to ﬂowering was
close to zero. Also, rate of progress to ﬂowering following 3-week treatment was
signiﬁcantly lower than ﬂowering rates of all treatments that induced complete
ﬂowering. Following treatment at 0 to 15 °C, the rate of progress to ﬂowering
continued to increase with durations up to 12 weeks, the longest duration tested
(Fig. 38). The minimum time to ﬂower and total time to ﬂower within each
duration occurred after 3 weeks at 5 °C, 5 weeks at 5 and 7.5 °C, 7 weeks at 2.5
to 7.5 °C, 9 weeks at 2.5 to 10 °C and 12 weeks at 2.5 to 12.5 °C (Fig. 3C and E).
Increasing the treatment durations at 0 °C and 5 to 15 °C for up to 12 weeks
reduced time to ﬂower (Fig. 3D). Following treatment at 2.5 °C, the average time
to ﬂower decreased for up to 9 weeks. The average total time to ﬂower varied
between 88 to 164 d, depending on the temperature and duration of treatment.
Minimum total time to ﬂower of treatments eliciting 275% ﬂowering was after 5

weeks at 0 to 7.5 °C and 7 weeks at 10 and 12.5 °C (Fig. 3 F).
Thermal Time to Flower. Average thermal time to ﬂower varied between 1226 to

3599 °C d, depending on treatment temperature and duration (Fig. 4A and B).

Minimum thermal time to ﬂower within each duration was recorded following

54

treatment for 5 weeks at 5 °C, 7 and 9 weeks at 0 to 5 °C and 12 weeks at 0 and
2.5 °C. Plants ﬂowering after treatment at 17.5 and 20 °C required signiﬁcantly

greater thermal time compared to plants treated at 0 to 12.5 °C at the respective
durations. Increasing treatment duration signiﬁcantly decreased thermal time to

ﬂower following treatment at 0 to 15 °C for 7 weeks.

Percent Reproductive Laterals. Treatment temperature, duration and their
interaction signiﬁcantly affected the percent reproductive laterals. Percent
reproductive laterals of all plants treated at 0 to 15 °C increased following up to 9
weeks treatment, while extending treatments to 12 weeks further increased
reproductive laterals on plants treated at 0, 5, 7.5, and 15 °C (Fig. 5A and B).
Highest percent reproductive laterals within each duration were produced
following treatment for 5 weeks at 7.5 °C, 7 weeks at 0 to 12.5 °C, 9 weeks at 2.5
°C and 12 weeks at 0 to 10 °C. The average percent reproductive laterals of the
two plants that ﬂowered following 17.5 and 20 °C treatments were 0.13 and 0.31,
respectively and were statistically similar to vegetative plants having no
reproductive laterals. Following treatments at 0 to 12.5 °C for 27 weeks, all
plants had >50% reproductive laterals. Although several individual plants
produced 100% reproductive laterals, no treatment combination elicited in 100%

reproductive laterals on all plants.

Number of Open Flowers. The number of open ﬂowers counted one week

following ﬁrst open ﬂower differed between the two replications, so data from the

55

two replications were analyzed separately. During both replications, treatment
temperature and duration and the interaction between them were signiﬁcant. In
both replications, plants that ﬂowered after 3 weeks of temperature treatments
produced signiﬁcantly fewer open ﬂowers compared to plants treated for 25
weeks (Fig. 6A). Overall, in both replications the number of open ﬂowers after 5-
week treatment at 0 to 12.5 °C was similar (Fig. 6B) and following 7-week
treatment, more ﬂowers were produced by plants treated at 7.5 to 12.5 °C
compared to the other temperatures (Fig. 60). Following 9 and 12-week
treatment, maximum number of flowers primarily occurred at 7.5 to 12.5 °C in
replication 1 whereas, in replication 2, plants treated at 5 to 12.5 °C generally
produced more ﬂowers (Fig. 60 and E). In replication 1 and 2, the number of
open ﬂowers following treatment at 0 °C did not increase with additional
treatment durations after 7 and 5 weeks, respectively. Number of open ﬂowers
after treatment at 2.5 to 7.5 °C increased as the treatment duration increased to
12 weeks in replication 1, while in replication 2, number of open ﬂowers after
treatment at 2.5, 5 and 7.5 °C did not increase after 9, 12 and 7 weeks,
respectively. The average number of open ﬂowers did not increase signiﬁcantly
after 7 and 9 weeks at 10 °C in replication 1 and 2, respectively. In both
replications, maximum open ﬂowers were produced after 9 weeks at 12.5 and 15

°C, with no further increase following treatment for 12 weeks.

Number of Flower buds and ﬂowers. Treatment temperature, duration and their

interaction had a signiﬁcant effect on number of ﬂower buds and ﬂowers

56

produced one week after ﬁrst open ﬂower on the 4‘“, 5th and 6th plant to ﬂower
within each treatment (Fig. 7A and B). The effect of treatment temperature on
the number of ﬂower buds and ﬂowers was highly variable and there was no
clear temperature optimum (Fig. 7A). Generally, increasing treatment duration
from 5 to 7 weeks increased the number of ﬂower buds and ﬂowers, while further
increases to 9 or 12 weeks had little to no effect on the number of ﬂower buds

and ﬂowers (Fig. 78).

Discussion

Campanula ‘Birch Hybrid’ has been previously reported to have an
obligate vemalization requirement (Enﬁeld, 2002; Niu et al., 2004). In the current
study only two out of 180 plants ﬂowered following exposure to 17.5 and 20 °C.
However, ﬂowering of these plants was delayed and their average rate of
progress to ﬂowering approached zero and was similar to vegetative plants.
Additionally, these two ﬂowering plants had few reproductive laterals and open
ﬂowers. These ﬁndings indicate that Campanula ‘Birch Hybrid’ has botanically a
near-obligate vemalization requirement and horticulturally, it can be
characterized as an obligate requirement necessary for complete, uniform, rapid
and profuse ﬂowering (Runkle et al., 2001). A similar vemalization response has
been reported in Oenothera fruticosa ‘Youngii-lapsley’ where only one out Of 180
plants ﬂowered in four different experiments, with considerably delayed ﬂowering

(Clough et al., 2001).

57

In this study, we established that, based on ﬂowering percentage, Tm," for
vernalizing Campanula ‘Birch Hybrid’ was <0 °C and Tmax was between 15 and
17.5 °C. The Tm," and Tmam for vemalization are species-speciﬁc and the reported
Tm," and Tmax of select species are presented in Table 1. Although some plants
exposed to -2.5 °C died or exhibited freezing injury, those that survived ﬂowered.
Based on this observation, we postulate that the Tm," of Campanula “Birch Hybrid’
is <-2.5 °C. The USDA Celd Hardiness Zones of the parents of Campanula
‘Birch Hybrid’, Campanula portenschlagiana Schult. and Campanula
poscharskyana Degen. are 4 and 3, respectively (Grifﬁths, 1994) and Campanula
‘Birch Hybrid’ is hardy at least to Zone 5 (personal observation). Direct transfer
of plants previously grown at 20 °C to -2.5 °C caused freezing injury and death
of some plants. It is likely that acclimation at 0 or 5 °C for two weeks prior to
treatment at -2.5 °C may have improved the survival of Campanula “Birch
Hybrid’ as reported by Engle (1994) for 20 species of herbaceous perennials
including Campanula carpatica.

The Tom for vemalization is species-speciﬁc and may differ depending on
the duration of temperature treatment and response being assessed (Lang,
1965). Additionally, the duration of low temperature treatment affects the kinetics
of the vemalization response, with increasing duration enhancing the
vemalization response until it saturates (Lang, 1965; Thomas and Vince-PrUe,
1984). In the current study, we investigated the vemalization response based on
the number of plants ﬂowering in a population, rate of progress to ﬂowering, time

to ﬂower from the end of temperature treatments, total time to ﬂower and thermal

58

time to ﬂower from start of temperature treatments to ﬂowering, percent
reproductive laterals, number of ﬂowers and number of ﬂower buds and ﬂowers.
When assessed as percentage of plants ﬂowering in the population, the Tom was
2.5 to 10 °C after 5 weeks and 0 to 12.5 °C after 27 weeks. Thus, there was an
interaction between treatment temperature and duration, and prolonging the
temperature treatments broadened the vemalization temperature optima of
Campanula ‘Birch Hybrid’. Since no treatment duration at 15 °C resulted in
100% ﬂowering, based on ﬂowering percentage, 12 weeks were insufﬁcient to
saturate the ﬂowering response at 15 °C.

The quantitative nature of the vemalization response is often illustrated by
ﬂowering time or leaf number at ﬂowering. Since the growthhabit of Campanula
‘Birch Hybrid’ did not permit monitoring leaf number at ﬂowering, only ﬂowering
time will be discussed. Time to ﬂower measured from the end of temperature
treatments at 0 °C and 5 to 15 °C decreased as treatment duration increased to
12 weeks. Tom for shortest ﬂowering time measured from end of treatments was
5 to 7.5 °C after 5 weeks, 2.5 to 7.5 °C after 7 weeks , 2.5 to 10 °C after 9 weeks
and 2.5 to 12.5 °C after 12 weeks. Hence, similar to ﬂowering percentage,
prolonging durations of treatments broadened the temperature optima of
ﬂowering response assessed as time to ﬂower, although the Top, for ﬂowering
percentage and time to ﬂower differed. The effect of thermoinductive
temperature and duration on total ﬂowering time measured from start of
temperature treatments was also evaluated to account for growing time during

the treatments. The Tom for total time to ﬂower and time to ﬂower was the same

59

but the duration at which the response saturated differed. Total time to ﬂower did
not decrease with additional treatment after 3 weeks at 0 °C, 7 weeks at 2.5 °C, 5
weeks at 5 and 7.5 °C and 7 weeks at 10 to 15 °C. It is noteworthy that 12-week
treatments at 0 to 15 °C signiﬁcantly increased total time to ﬂower compared to
5- to 9-week treatments, although time to ﬂower measured after treatments
decreased at 0 °C and 5 to 15 °C after 12 weeks. Thus, an additional 3 weeks of
treatment decreased time to ﬂower by <3 weeks.

The ﬂowering times reported in this study are up to 3 weeks longer than
those reported by Niu et al. (2004) and up to 3 weeks shorter than those reported
by Enﬁeld (2001). Differences in starting material and environmental conditions
prior to and following temperature treatments may have caused these differences.
Additionally, Niu et al. (2004) reported that temperature did not affect ﬂowering
time following treatment at 0 to 10 °C for 6 and 8 weeks, which is contradictory to
our ﬁndings and we can not explain this discrepancy.

The growth rate of plants is temperature-dependant and therefore, to
account for differences in plant development during temperature treatments, we
also evaluated ﬂowering response based on the thermal time to ﬂower, which
was computed from the start of temperature treatments. Thermal time to ﬂower
decreased following treatment at 0 to 15 °C for up to 7 weeks. The Top, based on
thermal time to ﬂower was 5 °C after 5 weeks, 0 to 5 °C after 7 and 9 weeks and
0 and 2.5 °C after 12 weeks. However, thermal time to ﬂower should
theoretically begin when a plant has made a developmental transition to

ﬂowering. Due to the lack of a physiological marker for this developmental switch,

60

thermal time to ﬂower is calculated from the start of temperature treatments and
therefore, may be overestimated. To evaluate the effect of this overestimation on
ﬂowering response, we chose 5 and 7 weeks as times when the switch to
ﬂowering may be made at all vernalizing temperatures and calculated the thermal
time after 5 and 7 weeks of temperature treatments. The effect of treatment
temperature and duration on thermal time to flower computed after 5- and 7-
week treatments was similar (Fig. 8A and B). Following treatment at 2.5 to 12.5
°C, the effect of treatment duration on thermal time to ﬂoWer computed after 5
and 7-week treatment was similar to thermal time to ﬂower from the start of
treatments. However, at 0 °C the minimum thermal time to ﬂower after 5- and 7-
week treatments was achieved after 12-week treatment. Tom at each treatment
duration varied slightly and was 5 or 7.5 °C after 5 weeks, 2.5 to 7.5 °C after 7
and 9 weeks and 0 to 7.5 °C after 12 weeks.

The effect of temperature treatments and durations on ﬂowering is often
evaluated based on ﬂowering time and ﬂowering time has been used to develop
vemalization models in crops including wheat and Easter lily (Lange and Heins,
1990; Rawson et al, 1998; Streck amd Schuh, 2005). Changes in vemalization
temperature optima based on the response variable being assessed were
reported in wheat cultivars by Rawson et al. (1998). The response assessed
altered the Top. of Campanula ‘Birch Hybrid' in this study and therefore, has
signiﬁcance in developing and interpreting vemalization models.

We also evaluated the effect of temperature treatments on percent

reproductive laterals, ﬂower number and number of ﬂower buds and ﬂowers. All

61

 

data were collected one week after the ﬁrst open ﬂower, which was arbitrarily
chosen as a time to account for gradual ﬂower opening. The Top. for percent
reproductive laterals were somewhat variable. Overall, Top. was 0 to 7.5 °C
following 5-week treatment and 0 to 12.5 °C following 7 to 12-week treatments.
Increasing treatment duration to 9 weeks typically increased percent reproductive
laterals. Overall, 27 weeks at 0 to 12.5 °C resulted in >60% reproductive laterals.
We expect that additional laterals were formed during the treatment at warmer
temperatures, and depending on the temperature and duration, only some of
these new laterals vernalized during the treatment. However, the speciﬁc
relationship of treatment temperature and duration with formation and
vemalization of laterals is unknown. Also, when ﬂowering was delayed,
additional vegetative laterals may have developed in the greenhouse, affecting
percent reproductive laterals reported. However, we found no correlation
between percent reproductive laterals and rate of progress to ﬂowering (data not
presented), indicating that their relationship is complex.

The number of open ﬂowers varied to even a greater extent with
signiﬁcant differences in ﬂower number between the two replications of the study.
Flowering response assessed as the number of open ﬂowers typically saturated
after 27 weeks of treatment depending on the temperature and replication.
Following a 7-week treatment at 0 to 12.5 °C, >25 open ﬂowers were present on
all ﬂowering plants. There was no consistent trend associated with the highest
number of ﬂower buds and ﬂowers at different treatment temperatures and

durations. This was at least in part due to high variability which could partly be

62

 

 

attributed to the small sample size. It is notable that 27 weeks at 0 to 12.5 °C
resulted in an average of 583 to 1261 ﬂower buds and ﬂowers. Unexpectedly,
there was no correlation between average number of ﬂower buds and ﬂowers
and average number of open ﬂowers (data not presented). The number of open
ﬂowers and ﬂower buds and ﬂowers may have been affected by percent
reproductive laterals and development of plants during temperature treatments,
especially at warmer temperatures although these relationships were unclear.
Number of open ﬂowers and ﬂower buds and ﬂowers inﬂuence marketability of
potted plants, however unlike edible crops, higher numbers do not always
translate into greater ﬁnancial returns. Following 27-week treatment at 0 to 12.5
°C Campanula ‘Birch Hybrid’ plants were marketable since the average number
of ﬂower buds and ﬂowers and open ﬂowers exceeded 500 and 25, respectively.
The complexity of vemalization is not only limited to its physiological
aspects, but also extends to the analysis and interpretation of the ﬂowering
responses. The effective treatment temperature and duration for optimum
ﬂowering response have been reported based on many responses including
ﬂowering percentage, node development, rate of node development, time to
visible bud or ﬂower, thermal time to ﬂower, number of ﬂower buds and ﬂowers at
ﬂowering and percent reproductive lateral nodes. In the present study we
conclusively demonstrate that the optimum temperature and duration for
vernalizing Campanula 'Birch Hybrid' vary signiﬁcantly based on the response
being assessed (Table 2 and 3). In general, temperatures between 0 and 12.5

°C successfully vernalized Campanula 'Birch Hybrid' when given for 27 weeks,

63

although extending durations for up to 12 weeks improved many ﬂowering
responses. We did not see any adverse effects of long-terrn storage of
Campanula 'Birch Hybrid' at 0 to 12.5 °C and therefore, when resources permit,

we recommend that commercial growers use longer durations.

64

Literature Cited

Atherton J.G., J. Craigon and EA. Basher. 1990. Flowering and bolting in carrot:
l. Juvenility, cardinal temperatures and thermal times for vemalization. J.
Hort. Sci. 65:423-429.

Brooking, LR. 1996. Temperature response of vemalization in wheat: a
developmental analysis. Ann. Botany. 78:507-512.

Chouard, P. 1960. Vernalization and its relations to dormancy. Annu. Rev. Plant
Physiol. 11:191-237.

Clough, E.A., A.C. Cameron, R.D. Heins and W.H. Carlson. 2001. Growth and
development of Oenothera fruticosa is inﬂuenced by vemalization duration,
photoperiod, forcing temperature, and plant growth regulators. J. Amer.
Soc. Hort. Sci. 126:269-274.

Enﬁeld, AL. 2002. Flower induction and cultural requirements for quick-cropping
of the herbaceous perennials Veronica spicata, Phlox paniculata,
Leucanthemum xsuperbum, Achillea, Gaura Iindheimen', and Campanula.
MS. Thesis, Michigan State University, East Lansing.

Engle, BE. 1994. Use of light and temperature for hardening of herbaceous
perennial plugs prior to storage at -2.5 °C. MS. Thesis, Michigan State
University, East Lansing.

Fausey, BA. 2005. the effects of light quantity and vernalization on growth and
ﬂowering of Arabidopsis, Achillea, Gaura, Isotoma, Lavandula and
Veronica. PhD Dissertation, Michigan State University, East Lansing.

Griffiths, M. 1994. Index of garden plants. p.200-201. Timber Press, Portland,
Ore.

Lang, A. 1965. Physiology of ﬂower initiation. In: Encyclopedia of plant
physiology (ed. W. Ruhland). p.1371-1576. Springer-Verlag, Berlin.

Lange, NE. and RD. Heins. 1990. Modeling temperature and photoperiod
inducted ﬂowering in Lilum longiﬂorum. Acta Hort. 272:115-119.

Lopez, RC. and Runkle ER. 2006. Temperature and photoperiod regulate
ﬂowering of potted Miltoniopsis orchids. HortScience. 41:593-597.

Niu, G., R. Heins, A. Cameron and W. Carlson. 2004. Vernalization and

devernalization of Campanula ‘Birch Hybrid’ and Leucanthemum
xsuperbum ‘Snowcap’. HortScience. 39:1647-1649.

65

Porter, JR. and M. Gawith. 1999. Temperature and the growth and development
of wheat: a review. European J. of Agron. 10:23-36.

Rawson, H.M, Zajac M. and LD. Penrose. 1998. Effect of seedling temperature
and its duration on development of wheat cultivars differing in
vemalization response. Field Crop. Res. 572289-300.

Roberts, E. and R. Summerﬁeld. 1987. Measurement and prediction of ﬂowering
in annual crops, p. 17-51. In: J.G. Atherton (ed.). Manipulation of ﬂowering.
Butterworth’s, London.

Robertson, M.J., Brooking LR. and J.T. Ritchie. 1996. Temperature response of
vemalization in wheat: modeling the effect on the ﬁnal number of
mainstem leaves. Ann. Botany. 78:371-381.

Runkle, E.R., R. Heins, A. Cameron and W. Carlson. 2001. Minireview of I
research activity: Horticultural ﬂowering of herbaceous perennials. '
Flowering Newsletter. 31 :34-43. 1,

Streck, NA 2003. A vemalization model in onion. Revista Brasileira de
Ag rociéncia. 10:99-105.

Streck, NA. and M. Schuh. 2005. Simulating the vemalization response of the
“Snow Queen” lily (Lilium Iongiﬂorum Thunb.). Scientia Agricola. 62:117-
121.

Suzuki A. and JD. Metzger. 2001. Vernalization in a greenhouse promotes and
synchronizes flowering of Osteospermum ecklonis Norl. HortScience.
36:658-660.

Thomas, B. and D. Vince-Prue. 1984. Juvenility, photoperiodism and

vemalization, p.408-439. In: M.B. Wilkins (ed). Advanced plant physiology.

Pitman Publ., London. ..
Vince-Prue, D. 1975. Photoperiodism in plants. p. 263-291. McGraw Hill, London.

Yan, W. and LA. Hunt. 1999. Reanalysis of vemalization data of wheat and
carrot. Ann. Botany. 84:615-619.

Yeh, D.M., Atherton J.G. and Craigon J. 1997. Manipulation of ﬂowering in

cineraria. Ill. Cardinal temperatures and thermal times for vemalization. J.
Hort. Sci. 72:379-387.

66

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Figure 1. Inﬂuence of temperature on node development of Campanula ‘Birch
Hybrid’ rooted cuttings. Open circles represent meaniSE and Solid lines
represent the regression equations generated using 722 observations from two
replications. The intercept and the slope of linear regression analysis for the rate
of node development were 0.0193i0.0023 and 0.00661: 0.0002, respectively.

 

Days per node
on A
O 01

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01

 

 

Rate (1Idays)
o .o
8 a o

.0
o
01

 

 

 

.0

O

O
p

 

O 5 1O 15 20

Temperature (°C)

70

Figure 2. Flowering percentage of Campanula ‘Birch Hybrid’ as a function of
treatment temperature (A) and duration (B). Flowering percentage was
computed after growing plants in a glass greenhouse set at 20 °C. Plants
without open ﬂowers after 105 d in the 20 °C greenhouse were considered
vegetative. Averages of pooled data from two replications are presented.

 

 

 

 

 

 

 

 

 

 

 

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Temperature (°C) Duration (weeks)
—e— 3weeks —O— 0°C
—-8— 5weeks ---l~-— 2.5 °C
—-wA-—- 7weeks -——A——- 5°C
v . Qweeks —"'— 7.50C
_. 9...- 12weeks ....... .....100C
—-—O—'— 12.5 °C
—-B"—' 15 0C
—-A— 17.5°C
---V--- 20°C

 

 

 

 

71

 

Figure 3. Inﬂuence of temperature treatment and its duration on rate of progress
to ﬂowering (A and B), time to ﬂower (C and D) and total time to ﬂower (E and F)
of Campanula ‘Birch Hybrid’. Rate of progress to ﬂowering was computed as
1/days to ﬁrst open ﬂower from end of temperature treatment. Time to ﬂower
was computed starting from end of temperature treatment and total time to ﬂower
was computed starting at the beginning of temperature treatments. MeanSiSE
data pooled from two replications are presented.

72

Rate of ﬂowering
Time to ﬂower (d) (1Id)

Total time to ﬂower (d)

 

 

 

 

5 10
Temperature (°C)

15 200

 

__e_
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5 weeks
7 weeks
9 weeks
12 weeks

 

 

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0 °C
2.5 °C
5 °C
7.5 °C
10 °C
12.5 °C

- 15°C

17.5 °C
20 °C

 

 

 

 

 

Figure 4. Inﬂuence of temperature treatment (A) and its duration (B) on thermal
time to ﬂower from beginning of treatment. Thermal time was calculated as
average daily temperature minus estimated leaf unfolding base temperature of
-2.9 °C and expressed in °C d. MeanSiSE of data pooled from two replications
are presented.

 

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3000 ~-

2500 .

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Thermal time to ﬂower (°C d)

 

 

 

 

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—e— 3 weeks —0— 0 °C
—-8— 5 weeks ——-I-—— 2.5 °C
—A— 7 weeks — + — 5 °C
—9— 9 weeks — -v — 7.5 °C
—e— 12 weeks -- O -- 10 °C

— —O— — 12.5 °C
— -El — 15 °C
— -A — 17.5 °C
— —v— — 20 °C

74

Figure 5. Percent reproductive laterals one week after first open ﬂower of
Campanula “Birch Hybrid’ as a function of treatment temperature (A) and duration
(B). Means:I:SE (A) and means (B) of 18 observations pooled from two
replications are presented.

 

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75

Figure 6. Inﬂuence of vemalization temperature on number of open ﬂowers
counted one week following ﬁrst open ﬂower in Campanula ‘Birch Hybrid’
vernalized for 3 weeks (A), 5 weeks (B), 7 weeks (C), 9 weeks (D) and 12 weeks
(E). Squares represent meantSE in replication 1 and diamonds represent
meaniSE in replication 2.

150?"‘

A1
100*

O-W—e—e—e—ev

Number of open ﬂowers

 

 

Temperature (°C)

76

Figure 7. Number of ﬂower buds and ﬂowers on 4‘", 5th and 6th plant to ﬂower
after ﬁrst open ﬂower, counted one week following the ﬁrst open ﬂower on
Campanula ‘Birch Hybrid’ plants as a function of (A) treatment temperature and
(B) treatment duration. MeanSiSE (A) and means (B) of 3 observations are
presented. Treatments resulting in <4 ﬂowering plants were considered to have
0 ﬂower buds and ﬂowers.

 

 

 

 

 

 

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Duration (weeks)

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-‘+>- 12weeks ...... 10.0 ,
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—-A— 175°C 1

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Figure 8. Themal time to ﬂower from 5 weeks (A) and 7 weeks (B) after
beginning of temperature treatment as a function of treatment temperature.
Thermal time was calculated 5 or 7 weeks from the beginning of temperature
treatment to the day of ﬁrst open ﬂower as average daily temperature minus
estimated leaf unfolding base temperature of —2.9 °C and expressed in °C d.
MeansiSE of data pooled from two replications are presented for treatment
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CHAPTER III:
DIANTHUS GRATIANOPOLITANUS ‘BATH’S PINK’ HAS A NEAR-OBLIGATE

VERNALIZATION REQUIREMENT

79

Dianthus gratianopolitanus ‘Bath’s Pink’ has a Near-Obligate Vernalization

Requirement

Sonali R. Padhye and Arthur C. Cameron

Department of Horticulture, Michigan State University, East Lansing, MI 48824

Additional index words. ﬂowering, herbaceous perennial, quantitative

vemalization response

Abstract

The ﬂowering response of Dianthus gratianopolitanus 'Bath's Pink' was
characterized following varying durations at vernalizing temperatures. Clonally
propagated plants were treated at 5 °C for 3, 6, 9, 12 or 15 weeks in experiment I,
at O, 5 or 10 °C for 2, 4, 6 or 8 weeks in experiment II and at O, 5, 10 or 15 °C for
1, 2, 4, 6 or 8 weeks in experiment Ill. Dianthus 'Bath's Pink' exhibited a
quantitative vemalization response following treatment at 0 to 10 °C and did not
vernalize at 15 °C in 8 weeks, the longest duration tested. Complete ﬂowering
was achieved following 24 weeks at 0 °C, 23 weeks at 5 °C and 8 weeks at 10 °C.
Based on time to anthesis and node number at anthesis, the ﬂowering response
was saturated following vemalization treatment at 0 °C for 24 weeks and 5 °C for
23 weeks. However, maximum ﬂower buds and ﬂowers at anthesis were

recorded following 8 weeks at 0 °C and 26 weeks at 5 °C. Plants took

80

signiﬁcantly longer to reach anthesis following 8-week treatment at 10 °C than 26
weeks at 0 °C and 24 weeks at 5 °C. Based on the minimum vemalization
duration required to achieve maximum ﬂowering response, the order of efﬁcacy

of vernalizing temperatures was 5 °C>0°C>>10°C.

Introduction

The wholesale value of herbaceous perennials has grown by 63% over
the last ﬁve years, reaching over $708 million in 2005 (US. Department of
Agriculture, 2006). This increase in sales of herbaceous perennials can in part
be attributed to the growing ability of producers to force and market perennials in
ﬂower at scheduled times. Forcing perennials in ﬂower requires an
understanding of the regulation of ﬂowering and subsequent manipulation of
environmental factors including photoperiod and vemalization to promote ﬂower
induction.

Flowering of many winter annuals, biennials and perennials is promoted
following exposure to low temperatures. This phenomenon, known as
vemalization, has been deﬁned as "the acquisition or acceleration of the ability to
ﬂower by a chilling treatment" (Chouard, 1960). Cold treatment that induces or
accelerates ﬂowering is also referred to as vemalization treatment (Napp-Zinn,
1987). An important aspect of vemalization is the temporal separation of the
thermoinductive treatment and the ﬂowering response. Therefore, at the end of
thermoinductive treatment, the only indicator of completion of vemalization

requirement is the subsequent ﬂowering after exposure to warm temperatures.

81

15

Insufﬁcient vemalization can result in incomplete and/or delayed ﬂowering, which
can cause considerable monetary losses to commercial producers of herbaceous
perennials.

Vernalization has been extensively studied in many winter annuals and
biennials and select perennials and the physiology of vemalization was
comprehensively reviewed decades ago (Chouard, 1960; Lang, 1965; Thomas
and Vince-Prue, 1984; Vince-Prue, 1975). Vernalization responses of plants can
be categorized as qualitative (obligate) or quantitative (facultative) (Lang, 1965).
Plants exhibiting a qualitative vemalization response require a cold treatment to
acquire the ability to ﬂower, whereas a cold treatment accelerates ﬂowering
and/or improves ﬂowering characteristics of plants with a quantitative
vemalization response. Vernalization has been shown to be a quantitative
process; the ﬂowering response increases with vemalization duration until
saturation (Lang, 1965). The effective ranges of thermoinductive temperatures
and durations that elicit a maximum vemalization response are species-speciﬁc.
For example, the thermoinductive temperatures producing the greatest ﬂowering
response of Veronica spicata L. 'Red Fox' and Isotoma axillan's Lindl. were -2.5
°C and 5 to 10 °C, respectively (Fausey, 2005). Therefore, selection of
appropriate thermoinductive temperatures is important for commercial production
of herbaceous perennials. Even within a species, the optimum temperature
range may change with changing durations (Lang, 1965). For example,
complete ﬂowering of Campanula “Birch Hybrid’ was achieved following a 5-week

vemalization treatment at 2.5 to 7.5 °C, however, all plants ﬂowered following a

82

 

 

9-week treatment at 0 to 12.5 °C (Chapter II). Hence, scheduling herbaceous
perennials in ﬂower necessitates empirical studies investigating the vemalization
temperatures and durations most effective for ﬂowering.

Dianthus gratianopolitanus is an herbaceous perennial native to western
and central Europe and is hardy to USDA Cold Hardiness Zone 3 (Grifﬁths,
1994). Dianthus 'Bath‘s Pink' is a selection of Dianthus gratianopolitanus popular
for its outstanding garden performance. Dianthus 'Bath's Pink' ﬂowers in mid-
spring in gardens in Michigan and is then largely vegetative through the summer
and autumn (personal observation). In a preliminary experiment, Dianthus
gratianopolitanus 'Bath's Pink' did not ﬂower without a vemalization treatment
when grown for 15 weeks in a greenhouse under 9- or 16-h photoperiods,
whereas all plants ﬂowered under 9- and 16-h photoperiods following a 15-week
treatment at 5 °C (Cameron et al., unpublished data). The objectives of this
study were to 1) characterize the ﬂowering response of Dianthus 'Bath's Pink' to
a range of thermoinductive temperatures and durations, and 2) determine the
effective vemalization temperatures and durations for complete, rapid and

uniform ﬂowering of Dianthus 'Bath's Pink'.

Materials and Methods

Propagation and Propagule Culture. Clonally propagated Dianthus ‘Bath’s Pink’
stockplants were maintained in a glass greenhouse set at 20 °C under a 16-h
photoperiod from 0600 to 2200 HR provided by a combination of sunlight and

high-pressure sodium (HPS) lamps. HPS lamps turned on when the ambient

83

light was below 200 pmol'm'z's'1 and turned off when ambient light was above
400 pmol'm"2's'1 and provided an additional 150 pmolm'z's'1 photosynthetic
photon ﬂux (PPF). Plants were hand watered when necessary with reverse
osmosis water containing nutrients (125 N, 12 P, 100 K, 65 Ca, 12 Mg, 1.0 Fe,
0.5 Mn, 1.0 Cu, 0.3 B, 0.1 Mo mg'L"; MSU Special, Greencare Fertilizers,
Chicago). Shoot-tip cuttings with 6 to 8 nodes were taken from stockplants and
dipped in a commercial rooting hormone (Dip ‘N Grow; Clackamas, Ore.)
containing 1 9L"1 indole—3-butyric acid and 0.5 g'L'1 naphthalene acetic acid, and
rooted in 72-cell trays (50-mL cell volume; Landmark Plastic Corporation, Akron,
Ohio) containing 50% commercial peat-perlite media (Sure-Mix, Michigan
Growers Products, Galesburg, Mich.) and 50% coarse perlite (T herm-O-Rock;
East Inc., New Eagle, Pa.). Cuttings were rooted under mist for ~2 weeks in a
propagation facility set at air and medium temperatures of 23 and 26 °C,
respectively and 0.3 kPa vapor pressure deﬁcit generated by injecting water
vapor in the air. After rooting, propagules were grown in 72-cell trays under the
same environmental conditions and nutritional regimen as stockplants until the

vemalization experiments were initiated.

Experiment I. Dianthus ‘Bath’s Pink’ cuttings were taken on 14 December 2003,
rooted for ~2 weeks and grown in 72-cell trays for ~1 month. The 72-cell trays
containing propagules were placed in a cooler set at 5 °C on 3 February 2004 for
3, 6, 9, 12 or 15 weeks. Cool-white ﬂuorescent lamps provided ~10 pmol'm’z's'1

PPF from 0800 to 1700 HR in the cooler. During vemalization treatments, plants

84

were hand watered when necessary with acidiﬁed well water (H2804 to a
titratable alkalinity of ~140 mg'L'1 CaC03) containing nutrients (40 N, 4 P, 40 K, 5
Ca, 0.3 Fe, 0.2 Mn, 0.2 Cu, 0.03 B, 0.03 Mo, 0.2 Zn mg'L“; MSU Special,
Greencare Fertilizers, Chicago). Ten additional plants were maintained in the

greenhouse as non-vernalized controls under the same conditions as stockplants.

Experiment II and III. DianthUs ‘Bath’s Pink’ cuttings were propagated on 27
April 2004, rooted for ~2 weeks in the propagation house and grown in 72-cell
trays for ~1 month in experiment II. On 11 June 2004, 72-cell trays containing
the propagules were placed in coolers set at 0, 5, or 10 °C for 2, 4, 6 or 8 weeks.
In experiment III, cuttings were taken on 27 October, 2004 and 1, 2, 4 and 6
weeks thereafter. Propagules were rooted for ~2 weeks, grown for 2 weeks and
placed in coolers set at 0, 5, 10 or 15 °C for 1, 2, 4, 6, or 8 weeks. The light and
nutritional conditions in the coolers were the same in all three experiments. An
additional 10 and 40 plants were maintained as non-vernalized controls in the

greenhouse in experiments II and Ill, respectively.

Growing Conditions and Environmental Control. In all three experiments,
following vemalization treatments, plants were transplanted into 13—cm square
plastic containers (1 .1-L volume) ﬁlled with commercial soil-less medium (Sure-
Mix; Michigan Grower Products, Galesburg, Mich.) and grown in a glass

greenhouse under the same light and nutritional regimens as stockplants.

85

 

 

 

The environmental conditions in the greenhouse were regulated by a
climate control computer (Priva, Model CD750; De Lier, The Netherlands). Type
E thermocouples (TT-E-40; Omega engineering, Stamford, Conn.) placed in
aspirated tubes measured temperature at plant height on every greenhouse
bench and the light intensity at plant height was measured by line quantum
sensors containing 10 photodiodes (Apogee Instruments, Logan, Utah) at two
locations in the greenhouse. Every 10 seconds, temperature and light data were
collected by the sensors connected to a CR10 datalogger (Campbell Scientiﬁc,
Logan, Utah) and hourly averages were computed and recorded in a computer.
The average daily temperatures (ADT) and average photosynthetic daily light
integrals (DLI) received by each plant were computed from the day of transplant
until the day of anthesis for all ﬂowering plants. For vegetative plants, ADT and
DLI were computed for 15 weeks from the date of transplant. Average ADT and

DLI of plants planted on the same date are reported in Table 1.

Data Collection and Analysis. At transplant, number of nodes was recorded and
at anthesis of ﬁrst ﬂower, date and number of nodes on the main axis were
recorded in all experiments. Additionally, number of ﬂower buds and ﬂowers at
anthesis were recorded in experiments II and III and number of vegetative and
reproductive laterals were recorded and percent reproductive laterals were
computed in experiment Ill. Plants without a visible flower bud 15 weeks after
transplant were considered vegetative. In all experiments, 10 plants were

subjected to each treatment combination and were arranged in a completely

86

randomized design. Data from experiment I were subjected to analysis of
variance using SAS’s PROC MIXED (SAS Institute, Cary, NC). Data from
experiments II and III were pooled for analysis as a factorial experiment with
vemalization temperature and duration as treatment factors using SAS’s PROC
MIXED. Least signiﬁcant difference procedure was used for paired comparisons

with P=0.05 as a maximum value for signiﬁcance.

Results and Discussion

Flowering response. In experiment I, 1 out of 10 non-vernalized control plants
ﬂowered and in subsequent experiments an average of 21% of non-vernalized
control plants ﬂowered (Fig. 1A and 2A). Thus, Dianthus ‘Bath’s Pink’ exhibited a
facultative vemalization response. Complete ﬂowering of Dianthus ‘Bath’s Pink’
was achieved following vemalization at 0 °C for 24 weeks, 5 °C for 23 weeks and
10 °C for 8 weeks. Only 1 out of 40 plants ﬂowered following vemalization
treatment at 15 °C for 58 weeks. The order of most effective vemalization
temperatures based on the shortest duration of vemalization treatment required
for achieving complete ﬂowering was 5 °C>0 °C>>10 °C.

The effective duration of vemalization for promotion of ﬂowering is
species-speciﬁc (Lang, 1965). For example, Tulipa species require a 14.5 to
23.5-week vemalization treatment (Dole and Wilkins, 2005), whereas radish can
be vernalized in 4 to 8 d (Erwin et al., 2002). However, many herbaceous
perennials including Campanula 'Birch Hybrid', Isotoma axillaris and Veronica

spicata 'Red Fox' require relatively longer (4 to 8 weeks) exposure to vernalizing

87

temperatures for complete ﬂowering (Fausey, 2005; Chapter II). In this study we
found that Dianthus 'Bath's Pink' ﬂowered completely after comparatively short
exposure to vernalizing temperatures and only 1, 2 and 23 weeks at 5 °C
resulted in 70, 95 and 100% ﬂowering, respectively (Fig. 2A). Similarly,
Oenothera fruticosa 'Youngii-lapsley' was completely vernalized following a 3-

week exposure to 5 °C (Clough et al., 2001).

Flowering Time. Vernalization promoted and synchronized ﬂowering of Dianthus
'Bath's Pink'. The time to reach anthesis of non-vernalized control plants was
highly variable. In experiment I, after 15 weeks in the greenhouse, only 1 out of
10 non-vernalized plants reached anthesis and in 114 d (Fig. 1B). In the
subsequent experiments, 21% of non-vernalized plants reached anthesis, in an
average of 58 d (Fig. 2B). Many species with a facultative vemalization response
ﬂower sporadically without vemalization treatment and the sporadic nature of
ﬂowering of Dianthus 'Bath's Pink' may explain the differences in ﬂowering times
of non-vernalized plants between the different experiments. We postulate that
plants that remained vegetative for 15 weeks in all experiments would have
subsequently ﬂowered at variable times and considerably increased the average
time to reach visible ﬂower bud and anthesis for the entire population.

In experiment I, compared to the non-vernalized controls, 3 weeks at 5 °C
accelerated ﬂowering signiﬁcantly and average time to subsequently reach
anthesis was 41 d. An additional 3 weeks of vemalization treatment hastened

ﬂowering time by approximately one week but vemalization treatment for up to 15

88

weeks did not further hasten ﬂowering. At 5 °C, the standard errors associated
with average time to anthesis were low after 23 weeks of vemalization treatment,
indicating that vemalization synchronized ﬂowering. Overall, the average
number of nodes on vernalized plants at anthesis did not vary from 3 to 15 weeks
of vemalization at 5 °C (Fig. 1C). Based on node number at planting and
anthesis, the reported 1 week decrease in ﬂowering time after 3- and 26-week
vemalization treatment was not due to developmental acceleration of ﬂowering.
Hence, this 1 week decrease in time to ﬂower can not be explained as the direct
effect of additional vemalization treatment. In experiment II and III, the ﬂowering
time of Dianthus 'Bath's Pink' decreased as vemalization duration at 5 °C
increased from 1 to 2 weeks, with no further reduction in time to ﬂower following
up to 8 weeks at 5 °C (Fig. 28). The number of nodes at anthesis followed a
trend similar to ﬂowering time (Fig. 2C). Overall, the results from all three
experiments indicate that complete and rapid ﬂowering of Dianthus 'Bath's Pink'
was achieved following vemalization treatment at 5 °C for 23 weeks.

Average time to reach anthesis and the average number of nodes at
anthesis signiﬁcantly decreased as the duration of vemalization treatment at 0 °C
increased to 4 weeks. Hence, complete and rapid flowering of Dianthus 'Bath's
Pink’ occurred following 24-week vemalization treatment at 0 °C. Dianthus
'Bath's Pink' ﬂowered completely and earlier following 4 weeks at 5 °C compared
to 4 weeks at 0 °C. However, 26 weeks at 0 °C or 5 °C were equally effective in

promoting ﬂowering based on ﬂowering percentage and ﬂowering time.

89

 

 

Vernalization treatment at 10 °C for 56 weeks resulted in incomplete
ﬂowering after 15 weeks of growing in the greenhouse, when we chose to
terminate the experiment. Given more time, it is possible that remaining plants in
these treatments may have ﬂowered, which would have increased average
ﬂowering time and node number than those reported here. An 8-week treatment
at 10 °C induced complete ﬂowering, however plants took signiﬁcantly longer to
reach anthesis and developed more nodes before anthesis compared to plants
vernalized at 0 °C for 26 weeks or 5 °C for 24 weeks. Hence, 0 and 5 °C were
more effective than 10 °C in promoting ﬂowering of Dianthus 'Bath's Pink'. Based
on ﬂowering percentage, ﬂowering time and node number at anthesis, Dianthus

'Bath's Pink' did not vernalize at 15 °C in 8 weeks.

Number of Flower Buds and Flowers and Percent Reproductive Laterals at
Anthesis. All treatments except 8 weeks at 0 °C and 26 weeks at 5 °C resulted in
similar numbers of ﬂower buds and ﬂowers at anthesis, and averaged between 3
to 9 (Fig. 2D). Plants averaged 16 to 21 ﬂower buds and ﬂowers following 8
weeks at 0 °C and 26 weeks at 5 °C. Since the DLIs received by plants under all
treatment combinations were similar, the increased ﬂower number was mostly a
result of vemalization response. Percent reproductive laterals at anthesis
averaged between 6 to 33 and followed the overall general trend of number of
ﬂower buds and ﬂowers at anthesis (Fig. 2E). Hence, these additional
vemalization durations contributed to improving the visual appeal of plants at

ﬂowering. Even after 8-week vemalization treatments at 0 or 5 °C, ﬂowering was

90

sparse and plants were of low quality. Since Dianthus 'Bath's Pink' produces
ﬂowers on the terminal and laterals that are developed at the onset of
vemalization, growing plants for a longer time prior to the initiation of
vemalization treatment could allow development of additional laterals prior to

vemalization and increase the number of ﬂowers produced.

Summary. Based on our results, Dianthus 'Bath's Pink' exhibited a near-obligate
vemalization response. The shortest vemalization duration resulting in complete
ﬂowering was 3 weeks at 5 °C although plants produced fewer ﬂower buds and
ﬂowers at anthesis compared to 26-week treatment. Following vemalization for 2
and 4 weeks, 5 °C was the most effective vemalization temperature based on
ﬂowering percentage and ﬂowering time, however after 26 weeks, 0 and 5 °C
were similarly effective. Dianthus 'Bath's Pink' ﬂowered completely after
vemalization at 10 °C only after 8-week treatment although ﬂowering was
delayed and fewer ﬂower buds and ﬂowers were produced at anthesis compared
to 8 weeks at 0 and 5 °C. Plants did not vernalize at 15 °C in 8 weeks and
therefore, the maximum temperature for vernalizing Dianthus 'Bath's Pink' is 210
°C. Compared to other treatments, more ﬂower buds and ﬂowers were produced
following 8-week vemalization at 0 °C and 26-week vemalization at 5 °C.
Additional growing of Dianthus ’Bath's Pink' prior to vernalization treatment could

improve ﬁnal plant quality.

91

Literature Cited

Chouard, P. 1960. Vernalization and its relations to dormancy. Annu. Rev. Plant
Physiol. 112191-237.

Clough, E.A., A.C. Cameron, R.D. Heins and W.H. Carlson. 2001. Growth and
development of Oenothera fruticosa is inﬂuenced by vemalization duration,
photoperiod, forcing temperature, and plant growth regulators. J. Amer.
Soc. Hort. Sci. 126:269-274.

Dole, J.M. and HF. Wilkins. 2005. Floriculture principles and species, p. 882-893.
Pearson Education Inc., Upper Saddle River, N.J.

Fausey, BA. 2005. The effects of light quantity and vemalization on growth and i
ﬂowering of Arabidopsis, Achillea, Gaura, Isotoma, Lavandula and , 5
Veronica. PhD Diss, Mich. State Univ., East Lansing. ]

Grifﬁths, M. 1994. Index of garden plants, p.363. Timber Press, Portland, Ore. . l2,

Napp-Zinn, K. 1987. vemalization - environmental and genetic regulation, p. 123-
132. In: J.G. Atherton (ed.). Manipulation of ﬂowering. Butterworths,
London.

Lang, A. 1965. Physiology of ﬂower initiation, p. 1371-1576. In: W. Ruhland (ed.).
Encyclopedia of plant physiology. Springer-Verlag, Berlin.

Thomas, B. and D. Vince-Prue. 1984. Juvenility, photoperiodism and
vemalization, p. 408-439. In: M.B. Wilkins (ed). Advanced plant physiology.
Pitman Publ., London.

US. Department of Agriculture. 2006. Floriculture and nursery crops 2005
summary. Agr. Stat. board, Wash., DC.

Vince-Prue, D. 1975. Vernalization, p. 263-291 In: Photoperiodism in plants.
McGraw Hill, London.

92

Table 1. Planting date, average daily temperature (ADT) and average
photosynthetic daily light integral (DLI) of Dianthus gratianopolitanus ‘Bath’s Pink’
vernalized for different durations. ADT and DLI were calculated for each
ﬂowering plant from day of transplant to anthesis and for vegetative plants, 105 d
after transplant. MeaniSE of ADT and DLI of 10, 30 and 240 plants are reported
for experiment 1, 2 and 3, respectively.

 

 

Experiment Vernalization Planting date ADT (°C) Avg. DLI
duration (week) (mol'm'z‘d’1)
1 0 2/03/2004 20.9 :I: 0.0 13.3 :I: 0.0
3 2/24/2004 20.7 :I: 0.0 13.6 :I: 0.2
6 3/16/2004 20.2 :t 0.0 17.5 :t 0.0
9 4/06/2004 20.8 :I: 0.1 14.4 :1: 0.5
12 4/27/2004 21.8 :I: 0.0 9.7 :l: 0.0
15 5/18/2004 22.4 1 0.0 10.5 :I: 0.0
2 0 6/11/2004 23.0 d: 0.0 11.6 d: 0.1
2 6/25/2004 23.0 :I: 0.1 12.0 :I: 0.1
4 7/09/2004 23.4 :I: 0.1 11.6 :I: 0.1
6 7/23/2004 22.3 :t 0.0 10.7 1 0.0
8 8/06/2004 22.6 :I: 0.0 10.4 :1: 0.0
3 0-8 1/08/2005 18.5 :I: 0.3 11.7 :I: 0.4

 

93

 

 

 

Figure 1. Flowering response of Dianthus gratianopolitanus ‘Bath’s Pink’ in
experiment I following vemalization at 5 °C at increasing durations. Open
symbols represent the averages for ﬂowering plants and vertical bars represent
standard errors. In A, ﬂowering percentage was computed 105 d after transplant.

 

 

 

 

   

 

 

 

 

 

   

 

 

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Vernalization duration (weeks)

94

 

Figure 2. Flowering response of Dianthus gratianopolitanus ‘Bath’s Pink’ in
experiment II and Ill following vemalization at 0, 5, 10 or 15 °C at increasing
durations. Open symbols represent the averages for ﬂowering plants and vertical

bars represent standard errors. In A, ﬂowering percentage was computed 105 d
after transplant. In D and E, data were collected at anthesis.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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95

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CHAPTER IV:
DAILY LIGHT INTEGRAL DURING SECONDARY INDUCTION AFFECTS
FLORAL EVOCATION IN COREOPSIS GRANDIFLORA 'SUNRAY'
FOLLOWING PRIMARY INDUCTION TREATMENTS OF VERNALIZATION OR

SHORT-DAY PHOTOPERIOD

96

Daily Light lntegral During Secondary Induction Affects Floral Evocation in
Coreopsis grandiﬂora 'Sunray' Following Primary Induction Treatments of

Vernalization or Short-day Photoperiod

Sonali R. Padhye, Erik S. Runkle and Arthur C. Cameron

Department of Horticulture, Michigan State University, East Lansing, MI 48824

Additional index words. dual induction, ﬂowering, herbaceous perennial,

photosynthetic photon ﬂux, short long day plant

Abstract

The inﬂuence of vemalization, photoperiod and photosynthetic daily light integral
(DLI) on ﬂoral evocation of Coreopsis grandiﬂora 'Sunray' was determined.
Primary induction treatments of vemalization or short days (SD) were provided to
clonally propagated Coreopsis 'Sunray' by either vernalizing plants at 5 °C under
long days (LD; 16-h photoperiod) or growing them under SD (9-h photoperiod) at
20 °C for O, 1, 2, 3, 4, 5, 6 or 8 weeks. Following the primary induction
treatments, plants were given secondary induction treatments of LD under a high
(~15 mol'm'z'd") or low (~6 mol'm’z‘d'1) DLI. Coreopsis 'Sunray' exhibited a
dual induction requirement for ﬂoral evocation, with primary induction by
vemalization or SD and secondary induction by LD. Vernalization was more

effective than SD in promoting ﬂoral evocation when their durations were sub-

97

dai-

 

optimal, as indicated by a higher ﬂowering percentage and accelerated ﬂowering.
In addition, following sub-optimal durations of primary induction treatment, ﬂoral
evocation of terminal and lateral buds was promoted under a high DLI in
secondary induction environment. Plants vernalized for sub-optimal durations
reached anthesis at an earlier developmental stage under high DLI. Hence, high
DLI partially substituted for the primary induction treatment of vemalization or SD.
Plant quality at anthesis was improved under high DLl by increased number of

inﬂorescences and reduction in plant height.

Introduction

Flowering of many species from temperate origins is regulated by environmental
cues of daylength (photoperiod) and low temperature to synchronize reproductive
development with favorable environmental conditions and to maximize
reproductive success. Flowering of photoperiodic species occurs, or is
accelerated when the photoperiod exceeds (long day (LD) plants) or is below
(short day (SD) plants) a critical value (Thomas and Vince-Prue, 1997). In some
species, ﬂoral evocation is promoted following exposure to low temperatures, a
phenomenon known as vemalization (Chouard, 1960). A low temperature
treatment can also be referred to as a vemalization treatment (Napp-Zinn, 1987).
In some species, a dual induction process regulates ﬂowering, which includes a
primary and a secondary induction phase (Heide, 1994). Flowering in several
temperate grasses and a few herbaceous perennial species is under dual

induction regulation; the primary induction is exposure to SD or vemalization and

98

 

 

the secondary induction is exposure to LD (Heide, 1994; Heide, 1995; Vince-
Prue, 1975; Wellensiek, 1960). Dual-induction requiring species with primary
induction by vemalization or SD have been categorized as vemalization-requiring
LD plants or short-long-day (SLD) plants, where vemalization can be replaced by
SD or vice versa (Vince-Prue, 1975).

Coreopsis grandiﬂora ‘Single Mayﬂeld Giant’ was reported to have a dual
induction regulation of ﬂowering, with primary induction by SD or vemalization
and secondary induction by LD (Ketellapper and Barbaro, 1966). Damann and
Lyons (1996) reported that the primary induction in Coreopsis grandiﬂora
'Sunray' occurred only by vemalization, and not by a SD treatment. However,
Runkle et al. (unpublished) found that Coreopsis 'Sunray' seedlings ﬂowered in
response to primary induction treatments of SD or vemalization.

The effect of DLI on ﬂoral evocation of herbaceous perennials is species-
speciﬂc. For example, when non-vernalized and sub-optimally vernalized Salvia
xsuperba Stapf 'Blaukonigin' was grown under a high DLI (14.4 mol'm’z'd"), a
greater percentage of plants flowered and ﬂowering was hastened compared to
plants grown under a low DLI (3.6 mol'm'z'd"; Waaseth et al., 2006). However,
after a saturating vemalization treatment, a higher DLI had no effect on ﬂowering
percentage and only slightly hastened ﬂowering (Waaseth et al., 2006). In
contrast, ﬂowering percentage and time to anthesis were not inﬂuenced by DLI
(from 5 to 20 mol'm'z‘d'1) in Achillea xmillefolium L. 'Red Velvet', Gaura
lindheimen' Engelm. & Gray 'Siskiyou Pink' and Lavandula angustifolia Mill.

'Hidcote Blue' (Fausey et al., 2005).

99

 

In our preliminary studies, following sub-optimal exposure durations of
vemalization or SD treatments, ﬂoral evocation was reduced under low
photosynthetic daily light integral (DLI) in the secondary induction environment
(Appendix A). The objectives of this study were to: 1) establish and characterize
the inﬂuence of primary induction treatments of SD or vemalization on ﬂoral
evocation in Coreopsis 'Sunray', and 2) evaluate the effect of DLI during
secondary LD-induction on ﬂoral evocation and subsequent ﬂowering

characteristics.

Materials and Methods

Stockplant Management and Propagule Culture. Coreopsis 'Sunray'
stockplants were developed by harvesting vegetative cuttings from a single
seedling selected for high vigor from a 50-seedling population. The cloned
stockplants were grown in 13-cm square plastic containers (1 .1-L volume) ﬁlled
with commercial soil-less medium (Sure-Mix; Michigan Grower Products,
Galesburg, Mich.) in a controlled environment chamber set at constant 20 °C
under a 16-h photoperiod (0600 to 2200 HR) provided by ﬂuorescent and
incandescent lamps that delivered ~150 umol'm”2's’1 photosynthetic photon ﬂux
(PPF) at plant height. Stockplants were watered when necessary with acidiﬁed
well water (H280; to a titratable alkalinity of ~140 mg'L’1 CaCOa) containing 40 N,
4 P, 40 K, 5 Ca, 0.3 Fe, 0.2 Mn, 0.2 Cu, 0.03 B, 0.03 Mo, 0.2 Zn mg'L'1 (MSU

Special, Greencare Fertilizers, Chicago). Shoot-tip cuttings with 34 nodes were

harvested from stockplants O, 1, 2, 3, 4, 5, 6 and 8 weeks prior to 1 March 2006.

100

 

The cuttings were dipped in a commercial rooting hormone (Dip 'n Grow; ;
Clackamas, Ore.) containing 1 9L"1 indole-3-butyric acid and 0.5 g'L’1
naphthalene acetic acid and rooted in 72-cell trays (50—mL cell volume;
Landmark Plastic Corporation, Akron, Ohio) containing 50% commercial peat-
perlite media (Sure-Mix, Michigan Growers Products, Galesburg, Mich.) and 50%
coarse perlite (Therm-O-Rock, East Inc., New Eagle, Pa.). Cuttings were rooted
under mist for 11 d and then weaned for 3 d by hand watering in a propagation
facility. During propagation, a 16—h photoperiod was provided by sunlight from
0600 to 1700 HR and incandescent lamps from 1700 to 2200 HR. The
propagation facility was set at air and medium temperatures of 23 and 26 °C,
respectively and a 0.3 kPa vapor pressure deﬁcit was maintained by injecting
water vapor in the air. After rooting, propagules were grown in 72-cell trays for 2
weeks in a controlled environment chamber under the same environmental and
nutritional conditions as stockplants until primary induction treatments were

initiated.

Primary Induction Treatments. For vemalization treatments, plants in 72-
cell trays were placed in a controlled environment chamber set at 5 °C under 16-
h photoperiod provided by a combination of ﬂuorescent and incandescent lamps
(~50 umol‘m'z's'1) from 0600 to 2200 HR. During vemalization, plants were
watered when necessary with the same nutrients as stockplants. Plants
receiving SD treatment were potted in ﬁlled 13-cm containers (as previously

described) and grown in a greenhouse under a 9-h photoperiod maintained by a

101

 

 

blackout system that opened between 0800 to 1700 HR. The photoperiod was

provided by sunlight supplemented by high-pressure sodium (HPS) lamps that

automatically turned on when ambient light was below 200 umol'm'z's'1 PPF and

ceased when ambient light exceeded 400 pmol'm’zs'1 PPF. HPS lamps

provided an additional 150 umol‘m'z's'1 PPF. In the greenhouse, plants were

watered using reverse osmosis water containing 125 N, 12 P, 100 K, 65 Ca, 12

Mg, 1.0 Fe, 0.5 Mn, 1.0 Cu, 0.3 B, 0.1 Mo mg'L'1 (MSU Special, Greencare ?
Fertilizers, Chicago). Induction treatments were imposed every 1, 2, 3, 4, 5, 6 :1 ~~..

and 8 weeks and constant LD (O-week primary induction treatment) and SD I

 

controls were planted such that all ended on 1 March 2006.

DLI Treatments during Secondary Induction. At the end of vemalization
treatments, plants were transplanted into 13-cm containers and maintained under
the same nutritional regimen as plants under SD. Plants from each treatment
combination were randomly selected and placed on greenhouse benches with a
high or low DLI. Based on preliminary experiments, 15 molm‘z'd’1 and 6
mol'm'z'd"1 were selected as target high and low DLIs for secondary induction,
respectively. The high DLI environment received 16-h photoperiod by sunlight
supplemented with HPS lamps (at 150 umol'm’z's‘1 PPF) when ambient light was
below 200 umol'm’z's‘1 PPF and ceasing when ambient light exceeded 400
umol'm‘z's’1 PPF. The low DLI environment was created by hanging permanent
woven shade curtain that reduced light transmission by 50% (OLS 50; Ludvig

Svensson, Charlotte, NC.) and applying a thin layer of whitewash on the glass

102

glazing. lrradiance at plant level was measured under high and low DLI

environments by line quantum sensors containing 10 photodiodes (Apogee

Instruments, Logan, Utah) connected to a CR10 datalogger (Campbell Scientiﬁc,

Logan, Utah). Light data were collected every 10 s and hourly averages were

recorded in a computer. Average DLI was computed for each plant from the day

of transplant until anthesis of ﬁrst inﬂorescence and 105 d from transplant for

ﬂowering and vegetative plants, respectively. Based on the measured DLI, '3
shading and lighting was manipulated such that average DLI received by all 2..

plants under high and low DLI environments ranged between 14.8 to 15.7 and

 

5.7 to 6.1 mol'm'z'd", respectively. The highest standard deviations for average
DLI received by a single plant were 5.3 and 2.1 under high and low DLI
environments, respectively.

Air temperature was measured every 10 s at plant height on each
greenhouse bench using Type E thermocouples ('lT-E-40; Omega engineering,
Stamford, Conn.) placed in aspirated tubes and hourly averages were recorded
in a computer. Average daily temperature (ADT) was computed for each plant for
the same durations as average DLI. ADT of plants under high and low DLI

environments ranged between 20.4 to 21.6 and 20.3 to 21.3 °C, respectively, and

 

the highest standard deviations for ADT received by a single plant was 1.7 and
2.0 °C under high and low DLI environments, respectively.

Data Collection and Analysis. The number of nodes on each plant was
recorded prior to primary and secondary induction treatments. At the

appearance of ﬁrst visible inﬂorescence and anthesis of ﬁrst inﬂorescence, dates

103

 

were recorded and time to visible inﬂorescence and anthesis were computed
from the initiation of secondary induction treatments. Plants without a visible
inﬂorescence 105 d after the start of secondary induction were considered
vegetative and ﬂowering percentage was computed for each treatment
combination. Rate of progress to anthesis was computed by taking the
reciprocal of time to anthesis; the rate of anthesis of vegetative plants was
reported as zero. The number of nodes below the terminal inﬂorescence was
also recorded at anthesis. At anthesis, the number of vegetative and
reproductive lateral shoots was counted and percent reproductive laterals was
calculated. Also, at anthesis, the number of inﬂorescences was recorded and
plant height from the plant base to the highest point was measured. Ten plants
were subjected to each treatment combination in a completely randomized
design. Response variables for each primary and secondary induction treatment
were individually subjected to regression analysis using Sigma Plot version 8.0

(SPSS Inc., Chicago) when signiﬁcant at P=0.0005.

Results

Flowering Response. No plants ﬂowered without a primary induction treatment
(Fig. 1A, E) or under continuous SD (data not shown). As primary induction
treatment duration increased, an increasingly greater percentage of plants
ﬂowered until all plants ﬂowered (Table 1). Under high DLI, 22 and 23-week
primary induction treatments of vemalization and SD induced complete ﬂowering,

respectively. Under low DLI, 0, 40, 70 and 100% plants flowered following 1, 2, 3

104

 

 

 

and 24-week vemalization treatments, respectively. When treated with SD for 1,
2, 3 and 24 weeks, 0, 0, 90 and 100% plants ﬂowered under low DLI,
respectively. Thus, following sub-optimal durations of primary induction
treatments, ﬂoral evocation was promoted under high DLI during secondary
induction. In addition, sub-optimal durations of vemalization were generally more
effective in promoting ﬂoral evocation than SD of the same duration for primary

induction.

Flowering Time. The rate of progress to anthesis increased with primary

 

induction treatment durations in similar patterns as did ﬂowering percentage (Fig.
18, F). Following sub-optimal durations of either primary induction treatment,
high DLI reduced time to anthesis (Fig, 10, G), and hence increased the rate of
anthesis. Vernalization was generally more effective in promoting ﬂoral
evocation than SD treatment following short durations irrespective of DLI during
secondary induction. Increasing the duration of vemalization treatment beyond 2
or 4 weeks did not further increase ﬂowering percentage under high and low DLIs
in the secondary induction environment, respectively, whereas, time to anthesis
decreased with increasing vemalization treatment duration up to 8 weeks.
Vernalization and SD treatment of 23 weeks caused similar decreases in time to
anthesis under both DLls . Following 8 weeks of primary induction by
vemalization or SD, plants ﬂowered in about 50 d. Average time to visible

inﬂorescence and anthesis of ﬁrst inﬂorescence was highly correlated for all

105

treatment combinations (Fig. 2A) and the time from visible inﬂorescence to
anthesis was ~23 d.

To evaluate the effect of induction treatments based on plant
developmental age, the number of nodes that developed during secondary
induction treatment to anthesis were assessed. Node number at anthesis
decreased with increasing primary induction treatment and followed a trend
similar to time to anthesis (Fig. 1D, H); there was a signiﬁcant correlation
between time to anthesis and node number at anthesis (Fig. 28). This suggests
that differences in time to anthesis could be primarily attributed to ﬂoral evocation
that occurred at an earlier developmental stage. On average, 7 nodes were
formed below the terminal ﬂower during secondary induction under both DLIs

after 8-week primary induction treatment.

Flowering Characteristics. Total laterals, percent reproductive laterals, number

of inﬂorescences and plant height at anthesis were measured to assess the
inﬂuence of induction treatments on the subsequent ﬂowering characteristics.

DLI during secondary induction did not affect the number of total laterals
produced (Fig. 3A, E). Vernalized ﬂowering plants produced 23 to 33 laterals

and there was no apparent relationship between total laterals and duration of
vemalization treatment. SD-treated plants produced 32 to 44 laterals on average,
and for treatments that induced complete ﬂowering, total lateral number

increased with exposure to SD.

106

 

 

Regression analyses of percent reproductive laterals yielded sigmoidal
response curves similar to ﬂowering percentage and rate of anthesis for all
induction treatment combinations (Fig. 3B, F). Under high DLI, vernalized plants
produced more reproductive laterals than SD-treated plants irrespective of
treatment duration. Under low DLI, plants vernalized for 25 weeks produced a
higher percentage of reproductive laterals than SD-treated plants. Most plants
had 100% reproductive laterals following vemalization treatments of 24 and 26
weeks under high and low DLls, respectively. However, with the exception of
one plant, SD treatments resulted in <100% reproductive laterals. Overall,
following SD treatment for 4 weeks, percent reproductive laterals averaged 77
and 66% under a high and low DLI, respectively.

The number of inﬂorescences increased with exposure duration of either
primary induction treatment (Fig. 3C, G). With longer primary induction treatment
durations, the number of inﬂorescences saturated after 2 and 4 weeks of
vemalization under a high and low DLI, respectively and after 4 weeks of SD
under low DLI. However, under a high DLI, the number of inﬂorescences on SD-
treated plants increased following primary treatment for up to 8 weeks, the
longest duration tested. Typically, following either primary induction treatment,
more inﬂorescences were produced under a high than low DLI.

With few exceptions, plants under the high DLI during secondary induction
were shorter at anthesis compared to plants grown under the low DLI (Fig. 3D,

H). Generally, duration of either primary treatment did not affect plant height at

107

 

 

 

anthesis and plants were of similar height following either primary induction

treatment.

Discussion

Coreopsis 'Sunray' responded qualitatively to dual induction treatments
and primary induction was achieved by vemalization or SD. These results
contradict the ﬁnding of Damann and Lyons (1993) that SD can not substitute for
vemalization as a primary induction treatment in Coreopsis 'Sunray'. Damann
and Lyons (1993) transferred seedlings to LD (natural photoperiod with 4-h night
interruption) after they had unfolded up to 12 nodes (according to Yuan et al.
(1997), juvenility ends at ~8 nodes) under SD (9-h photoperiod) and observed no
ﬂowering. Although Damann and Lyons (1993) did not specify the duration of SD
exposure, in our experiment, ~1 node was unfolded in 1 week under SD at 20 °C
(data not shown). Hence, it is probable that the SD exposure provided by
Damann and Lyons was suboptimal. Damann and Lyons (1933) did not indicate
the DLI during the secondary induction and it is possible that secondary induction
was under low DLI or that there were other limiting factors. Our results indicate
that in Coreopsis 'Sunray', following sub-optimal durations of primary induction
treatment, ﬂoral evocation can be reduced or inhibited under low DLI during the
secondary induction and this may explain why our results differed from those of
Damann and Lyons.

Niu et al. (2002) reported 0% ﬂowering of Coreopsis 'Sunray' seedlings

under LD (16-h photoperiod; ~3.7 mol'm‘z‘d’1 DLI) following a 5-week exposure

108

 

 

to SD (12-h photoperiod). We have observed 0% and 50% ﬂowering following
SD treatment at 12-h photoperiod, when secondary induction was under low (9.6
mol'm'z'd") and high (16.6 mol'm'z'd'1) DLls, respectively (Appendix B).
Therefore, the lack of ﬂowering after the 5-week SD treatment reported by Niu et
al. (2002) could be attributed to the photoperiod used.

Damann and Lyons (1996) reported 20% ﬂowering under continuous SD
(9-h photoperiod), whereas in the current study, no plants ﬂowered under
constant SD. We can not explain this discrepancy. Although our results are
based on clonally propagated plants developed from a single seedling, Runkle et
al. (unpublished) found a similar ﬂowering response in Coreopsis 'Sunray'
seedlings. Our results are consistent with the ﬂowering response of Coreopsis
grandiﬂora 'Single Mayﬁeld Giant', which was reported to have a similar dual
induction requirement (Ketellapper and Barbaro, 1966). For both Coreopsis
grandiﬂora cultivars, the primary induction requirement could be met by either SD
or vemalization, and the secondary induction requirement by LD.

Dual regulation of ﬂowering by SD or vemalization treatment followed by
LD has been reported in only a few herbaceous perennial species including
Campanula medium L. and Leucanthemum vulgare Lam. (Heide, 1995;
Wellensiek, 1960) and several grasses of temperate origin (Heide, 1994).
Initially, it was proposed that in Campanula medium, primary induction via
vemalization or SD occurs through distinct pathways (Wellensiek et al., 1960).
Recently, Dubcovsky et al. (2006) reported that in dual induction-requiring

Triticum monococcum, exposure to SD downregulated the vemalization

109

 

 

responsive ﬂowering inhibitor VRN2 and during secondary induction under LD,
the meristem identity gene VRN1 was upregulated. Hence, in Triticum
monococcum, SD substituted for vemalization by regulating upstream
vemalization responsive genes. Although the molecular regulation of ﬂoral
evocation in response to primary induction via vemalization or SD in dual
induction requiring herbaceous perennials including Coreopsis 'Sunray' remains
unknown, a mechanism similar to Triticum monococcum may be responsible for
the unique ﬂowering requirement.

Following sub-optimal durations of primary induction treatment,
vemalization at 5 °C under LD was typically more effective in promoting ﬂoral
evocation in Coreopsis 'Sunray' than the same durations of SD treatment at 20
°C based on ﬂowering percentage, ﬂowering time, percent reproductive laterals
and number of inﬂorescences at anthesis. Hence, it appears that Coreopsis
'Sunray' was more sensitive to primary induction via vemalization than SD
treatment under our experimental conditions. Heide (1995) reported that in
Leucanthemum vulgare, primary induction predominantly occurred through
vemalization and SD treatment did not fully substitute for vemalization. Also,
Campanula medium ﬂowered completely when primary induction was by
vemalization whereas, after primary induction by SD treatment, ﬂowering
percentage was rarely 100% (Wellensiek, 1960). These results indicate that
vemalization has a stronger induction effect than SD treatment for plants with a
dual induction ﬂowering including Coreopsis 'Sunray', Leucanthemum vulgare

and Campanula medium.

110

 

 

 

 

 

”——

DLI before, during, and after induction treatments can inﬂuence ﬂoral
evocation in some herbaceous perennial species. For example, low pre-
vernalization DLI (4 mol'm"2'd") induced a higher ﬂowering percentage in
Lavandula angustifolia 'Hidcote' and hastened ﬂoral evocation in Aquilegia
xhybrida Sims 'Remembrance' compared to high DLI (14 mol'm'z'd'1; Niu et al.,
2002). Although the inﬂuence of DU prior to primary induction treatments on
ﬂoral evocation was not evaluated in this study, Niu et al. (2002) reported that
pre-vernalization DLI did not affect ﬂoral evocation or ﬂowering characteristics of

Coreopsis 'Sunray'. In the current study, following 53-week exposure to either

 

primary induction treatment, fewer plants of Coreopsis 'Sunray' ﬂowered during
secondary induction under a low DLI, compared to a high DLI.

We also assessed the inﬂuence of DLI during secondary induction on
timing of ﬂoral evocation. The reported time to anthesis of Coreopsis 'Sunray'
after saturating durations of either primary induction treatment in this study is
consistent with previous reports (Damann and Lyons, 1996; Yuan et al., 1997;
Yuan et al., 1998). Following sub-optimal vemalization treatment, ﬂowering was
hastened under a high DLI due to ﬂoral evocation at an earlier developmental
stage as indicated by the formation of fewer nodes under the terminal ﬂower

compared to plants under the low DLI. However, for treatment durations that

 

resulted in complete ﬂowering, SD treatment resulted in a similar number of
nodes below the terminal ﬂower under both DLls. Therefore, at least in the case
of plants vernalized for sub-optimal durations, high DLI was a promoter of ﬂoral

evocaﬁon.

111

In many determinate herbaceous perennial species including Coreopsis
'Sunray', the strength of the induction "signal" can be assessed by evaluating
percent reproductive laterals (Fausey, 2005; Heide, 1995) since, in these species,
the terminal bud is induced ﬁrst, followed by the lateral buds, from apical to
basipital (personal observation). Coreopsis 'Sunray' produced a similar number
of total laterals at anthesis, although under high DLI, there were more
reproductive laterals following vemalization treatment for S5 weeks and following
all durations except 4 weeks of SD treatment. Hence, the observed decrease in
percent reproductive laterals under low DLI was due to failure of some laterals to
become reproductive. Overall, our results suggest that following sub-optimal
exposure to primary induction treatments, ﬂoral evocation of apical and basipital
buds was limited and ﬂoral evocation was delayed during secondary induction
under low DLI.

A similar role of DLI has been reported in plants with a quantitative
vemalization response including Salvia xsuperba 'Blaukonigin' (Waaseth et al.,
2006) and Digitalis purpurea L. 'Foxy' (Fausey et al., 2001). Several
physiological studies have investigated the mechanism involved in promotion of
ﬂoral evocation by high DLI and it has been proposed that the increased
concentration of assimilates under high DLI may promote ﬂoral evocation via a
feed fonNard control and the timing of elevated assimilate supply may also be of
signiﬁcance (reviewed by Sachs, 1987). Waaseth et al. (2006) postulated that
regulation of ﬂowering occurred in Salvia xsuperba 'Blaukonigin' via a PPF-

dependant ﬂowering pathway and proposed that following vemalization the PPF-

112

 

 

I...

 

 

 

dependant ﬂowering pathway became redundant. However, in the current study,

 

the lack of ﬂowering without an induction treatment under high DLI does not
support the existence of PPF-dependant ﬂowering pathway operating parallel to
a primary induction pathway in Coreopsis 'Sunray'. Alternatively, Coreopsis
'Sunray' requires at least some primary induction for PPF-dependant pathway to
operate, implying that high DLI can partially substitute for primary induction
treatments of vemalization or SD. 7 i
The observed increase in number of total laterals in Coreopsis 'Sunray' I J

plants treated with SD for 23 weeks was probably due to the formation of

 

additional laterals during SD treatment, and since some of these laterals did not
receive sufﬁcient SD exposure, percent reproductive laterals did not increase
with increasing durations of SD treatment. Due to development of laterals under
SD that were not induced by the end of SD treatment, percent reproductive
laterals of SD-treated plants never reached 100%, unlike vernalized plants. Also,
plants treated with 1 or 2 weeks of SD had more laterals than plants treated for 3
weeks as their ﬂowering was delayed and therefore additional laterals may have
been developed during secondary induction.

In addition to promoting ﬂoral evocation and its timing, high DLI may also

impact various ﬂowering characteristics including number of laterals and

 

inﬂorescences produced and plant height at anthesis. As discussed earlier, DLI
during secondary induction of Coreopsis 'Sunray’ did not affect the number of
laterals produced at anthesis. However, the number of laterals increased with

DLl in three Chrysanthemum cultivars (Schoellhorn et al., 1996) and in Heliconia

113

 

'Golden Torch' (Catley and Brooking, 1996). In this study, Coreopsis 'Sunray'
typically had more inﬂorescences at anthesis under high DLI compared to low
DLI. This is consistent with production of more inﬂorescences under higher DLl
reported in several herbaceous annual and perennial species, presumably due to
availability more assimilates for reproductive development (Fausey et al., 2005;
Faust et al., 2005; Niu et al., 2001; Pramuk and Runkle, 2005). The effect of DLI

on plant height is species-speciﬁc, with high DLI promoting, inhibiting or not

. ‘r'IA-A‘..“

affecting stern extension, depending on the species (Faust et al., 2005). : 3.

Generally, under high DLI, stem extension of Coreopsis 'Sunray' plants was

 

inhibited compared to low DLI.
In summary, our results demonstrate that ﬂowering of Coreopsis 'Sunray'

is regulated by a dual induction process, with primary induction via vemalization

 

or SD treatment and the secondary induction via LD. Hence, Coreopsis 'Sunray'
can be categorized as a short-long-day plant or vemalization-requiring LD plant.
Vernalization was somewhat more effective in promoting ﬂoral evocation if
induction treatment durations were sub-optimal. Floral evocation of Coreopsis
'Sunray' was promoted by high DLl during secondary induction following

exposure to sub-saturating durations of either primary induction treatment. High

 

DLl during secondary induction also improved plant quality: inﬂorescences
produced at anthesis were greater and plants were shorter when grown under a

high DLI.

114

 

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Vince-Prue, D. 1975. Vernalization, p.263—291. In: Photoperiodism in plants.
McGraw-Hill, London, New York.

Waaseth, G., R. Moe, R.D. Heins and 8.0. Grimstad. 2006. Effect of
photosynthetic photon ﬂux and temperature on ﬂoral evocation and
development in the vemalization-sensitive ornamental perennial Salvia
xsuperba 'Blaukonigin'. J. Amer. Soc. Hort. Sci. 1312437444.

116

Wellensiek, SJ. 1960. Flower formation in Campanula medium. Meded.
Landbouwhogeschool, Wageningen. 60: 1-18.

 

 

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118

Figure 1. Flowering of Coreopsis grandiﬂora 'Sunray' in response to primary
induction treatment of vemalization (O/El) at 5 °C with 16—h long days (A-D) or
short-days (SD; A/V) under 9-h photoperiod at 20 °C (E-H) for varying durations
and secondary induction treatments of long days under high (O/A) or low (El/V)
daily light integrals (DLI). Flowering response was assessed as ﬂowering
percentage 105 cl from initiation of secondary induction (A, E), rate of progress to
ﬂowering computed as reciprocal of time to anthesis with ﬂowering rate of
vegetative plants reported as zero (B, F), time to anthesis from initiation of
secondary induction (C, G) and number of nodes from initiation of secondary
induction until anthesis (D, H). Closed symbols are based on 10 observations
and open symbols represent averagetSE of 10 replicates for B. F and
averageztSE of ﬂowering plants for CD, G-H. Solid and dashed lines represent
regression analyses for individual plants under high and low DLls, respectively.

119

Rate of
anthesis (1Id)

Flowering percentage

0

Nodes (no.)

Time to anthesis (d)

Vernalization

SD treatment

 

100 . ..
75 ..
50 0
25 0

 

 

 

.0 .0
O O
O .3
\l 01
01 0

0.0000 v“

 

 

 

7

 

 

 

 

 

 

2 4 6

8

0

2 4 6

Primary induction treatment duration (weeks)

120

 

 

 

Figure 2. Correlation analysis between time to anthesis and time to visible
inﬂorescence of Coreopsis grandiﬂora 'Sunray' plants computed from the start of
secondary induction to anthesis and appearance of ﬁrst visible inﬂorescence,
respectively (A). Correlation analysis between time to anthesis and number of
nodes developed from the start of secondary induction to anthesis (B). Data are
pooled for all ﬂowering plants that were previously given primary induction
treatments of vemalization (16-h photoperiod at 5 °C) or short-days (9-h
photoperiod at 20 °C). Each regression line islgenerated usin over 225
observations for all ﬂowering plants (signiﬁcant at P=0.0001; =0.96 and 0.79 for
A and B, respectively).

 

 

 

 

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40 50 60 70 80 90

Time to anthesis (d)

121

 

 

 

Figure 3. Flowering characteristics of Coreopsis grandiﬂora 'Sunray' in response
to primary induction treatment of vemalization (<>/l:l) at 5 °C with 16-h long days
(AD) or short-days (SD; A/V) under 9-h photoperiod at 20 °C (E-H) at varying
durations and secondary induction treatments of long days under high (<>/A) or
low (El/V) daily light integrals (DLI). Flowering characteristics assessed included
total laterals (A, E), percent reproductive laterals (B, F), number of inﬂorescences
(C, G) and plant height (D, H) at anthesis. Plant height was measured from plant
base to the highest point. Open symbols represent averagetSE of 10 replicates
for B-C, F-G and averageiSE of ﬂowering plants for A, D, E, H. Solid and
dashed lines represent regression analyses for individual plants under high and
low DLls, respectively.

 

122

Total
laterals (no.)

Percent

reproductive laterals

lnﬂorescences

(no.)

Height (cm)

50

Vernalization

SD treatment

 

20 ..

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6

 

APPENDIX A:
DAILY LIGHT INTEGRAL AFFECTS THE FLOWERING RESPONSE OF
COREOPSIS GRANDIFLORA 'SUNRAY' TO INDUCTIVE TREATMENTS OF

VERNALIZATION OR SHORT-DAY PHOTOPERIOD

124

 

 

 

 

 

Daily Light Integral Affects the Flowering Response of Coreopsis grandiﬂora

'Sunray' to Inductive Treatments of Vernalization or Short-day Photoperiod

Introduction
Coreopsis grandiﬂora ‘Sunray’ ﬂowers under long-day (LD) photoperiods

following vemalization (Yuan et al., 1998). Short-day (SD) photoperiods

 

 

substituted for vemalization in Coreopsis ‘Single Mayﬁeld Giant’ (Ketellapper and

Barbaro, 1966). Damann and Lyons (1993) reported that Coreopsis ‘Sunray’ l‘

 

seedlings did not ﬂower in response to 10 weeks of SD followed by LD. However,
Runkle et al. (unpublished data) found that 10 weeks of SD followed by LD
resulted in complete (100%) ﬂowering of Coreopsis ‘Sunray’. The objectives of
this study were to: 1) establish Coreopsis ‘Sunray’ as a short-Iong-day (SLD)
plant, 2) determine the effective duration of vemalization or SD treatment prior to
LD for complete and uniform ﬂowering, and 3) determine whether photoperiod

during vemalization inﬂuences ﬂowering.

Materials and Methods

Stockplant Management and Propagule Culture. To minimize genetic variability, __
stockplants were developed from a single seedling and all plants used in

subsequent experiments were clonally propagated. The plant selection for clonal

propagation was made from a seedling population based on high vigor.

Stockplants were maintained in 13-cm square pots (1.1 L) in commercial soil-less

125

 

media containing peat and perlite and grown under a 16-h photoperiod at a 20 °C

temperature setpoint in a glass greenhouse and growth chamber in experiments I

 

and ll, respectively. In the greenhouse, the 16-h photoperiod was provided from
0600 to 2200 HR by a combination of sunlight and high-pressure sodium (HPS)
lamps that turned on when ambient light levels were below 200 umol'm'z's’1
photosynthetic photon ﬂux (PPF) and ceased when the light exceeded 400

pmol'm'1's'1 PPF. The HPS lamps provided an additional 150 pmorm‘z-s" PPF.

 

In the growth chamber, the 16-h photoperiod was provided by a combination of

ﬂuorescent and incandescent lamps and the PPF was ~150 umol'm'z's".

 

Stockplants grown in the greenhouse were watered with reverse osmosis water
containing 125 N, 12 P, 100 K, 65 Ca, 12 Mg, 1.0 Fe, 0.5 Mn, 1.0 Cu, 0.3 B, 0.1
Mo mg'L'1 (MSU Special, Greencare Fertilizers, Chicago). In the growth
chamber, stockplants were watered with acidiﬁed well water (H2804 to a
titratable alkalinity of ~140 mg‘L’1 CaCOa) containing nutrients (40 N, 4 P, 40 K, 5
Ca, 0.3 Fe, 0.2 Mn, 0.2 Cu, 0.03 B, 0.03 M0, 0.2 Zn mg'L"; MSU Special,
Greencare Fertilizers, Chicago).

In experiment I, cuttings were taken on 27 February 2004, dipped in a
commercial rooting hormone (Dip ‘N Grow; Clackamas, Ore.) containing 1000

ppm indole-3-butyric acid and 500 ppm naphthalene acetic acid and stuck in 72-

 

cell trays (50-mL cell volume; Landmark Plastic Corporation, Akron, Chic)
containing 50% commercial peat-perlite media (Sure-Mix, Michigan Growers
Products, Galesburg, Mich.) and 50% coarse perlite (T herm-O-Rock; East Inc.,

New Eagle, Pa.). Cuttings were rooted under mist in a propagation house under

126

 

16-h photoperiod from 0600 to 2200 HR, provided by a combination of sunlight
and incandescent lamps. The air and soil temperature setpoints in the
propagation house were 23 and 26 °C, respectively and a 0.3 kPa vapor
pressure deﬁcit was generated by injecting water vapor in the air. Plants were
rooted for 2 weeks and then grown in 72-cell trays in a growth chamber for 2
weeks under the same environmental and nutritional conditions as stockplants
grown in the growth chamber. In experiment ll, cuttings were taken 0, 4, 8, 16,
20 and 42 d prior to 6 December 2004 and were rooted and grown as in

experiment I.

Inductive Treatments. In experiment I, on April 6 2004, a group of plants (4
weeks old from the stick date) was vernalized by placing cell trays in a cooler at 5
°C under ~100 umol'm’z‘s’1 PPF for 16-h provided by a combination of
ﬂuorescent and incandescent lamps from 0600 to 2200 HR. A second group of
plants was potted in 13—cm square pots containing the same media as
stockplants and was given a short-day treatment in the greenhouse set at 20 °C
by placing them under a 9-h photoperiod provided by a blackout system that
opened at 0800 HR and closed at 1700 HR. During the 9-h photoperiod, light
intensity was controlled similar to the stockplants grown in the greenhouse. In
experiment ll, 4, 8, 12, 16, 20 and 42 d prior to 6 December 2004 plants were
either given vemalization or short-day treatment as described above. In both
experiments, one additional group of plants was maintained in the greenhouse in

13-cm square pots under a 16-h photoperiod as long-day controls and another

127

 

 

 

 

group of plants was maintained under a 9-h photoperiod as short-day controls.
The inductive treatments were provided to nine plants per treatment combination
for 14, 28, 42 or 56 d in experiment I and 10 plants per treatment combination for

4, 8, 12, 16, 20 or 42 d in experiment ll.

Plant Culture and Climate Control. Following the vemalization treatments, plants

were potted in 13-cm square pots and grown in the greenhouse at 20 °C setpoint

 

temperature and 16-h photoperiod similar to the greenhouse-grown stockplants

along with plants treated with SD. The greenhouse climate was controlled by a

 

climate control computer (Priva, Model CD750; De Lier, The Netherlands). Air
temperature was measured on each greenhouse bench by type E thermocouples
(TT-E-40; Omega engineering, Stamford, Conn.) placed in aspirated tubes and
PPF at plant height was measured at two locations by line quantum sensors
containing 10 photodiodes (Apogee Instruments, Logan, Utah). Temperature
and light sensors were connected to a CR10 datalogger (Campbell Scientiﬁc,
Logan, Utah) and data were collected every 10 s and hourly averages were

calculated and recorded in a computer.

Data Collection. The number of nodes on each plant was recorded prior to

 

initiation of inductive treatments in both experiments and again at the end of
inductive treatments in experiment II. On the day of ﬁrst visible inﬂorescence and
anthesis of ﬁrst inﬂorescence, dates were recorded and time to visible

inﬂorescence and anthesis was computed starting from the end of inductive

128

treatments. At anthesis, number of inﬂorescences and lateral shoots were
counted and plant height was measured from the media surface to the highest
point. Plants without inﬂorescene 105 d after the start of forcing were considered
vegetative. Average daily temperature (ADT) and daily light integral (DLI) were
computed for each plant from the start of forcing until anthesis and 105 d for

ﬂowering and vegetative plants, respectively, and are presented in Fig. 1.

Results and Discussion

The Eﬁ'ect of Inductive Treatments on Flowering. No SD control plants
ﬂowered in either experiment (data not shown) and no LD control plants ﬂowered
in experiment ll (Fig. 2A and G). In experiment I, two of the nine LD control
plants ﬂowered, although the ﬂowering plants were considerably delayed
compared to plants that had received inductive treatments (Fig ZB, C, H, l).
Additionally, these two ﬂowering LD control plants developed an average of only
2 inﬂorescences (Fig. 2D and J) and were considerably shorter compared to
plants that received inductive treatments (Fig. 2F and L). All plants ﬂowered
following exposure to 5 °C or SD for 214 d in experiment I, indicating that
Coreopsis 'Sunray' has a near-obligate vemalization requirement that can be
substituted by a SD treatment prior to exposure to LD. Therefore, Coreopsis

'Sunray' can be considered as a vemalization-requiring or SLD plant.

Effective Durations of Inductive Treatments. Fourteen d of vemalization or

SD was sufﬁcient to induce complete ﬂowering in experiment I, but in experiment

129

, ¢-...

 

 

 

ll, complete ﬂowering was achieved only after 216 d vemalization and 42 d SD
treatment; shorter durations of inductive treatments induced incomplete ﬂowering.
The cultural conditions were identical in the two experiments, although the
forcing environments were different as indicated by the differences in ADT and
DLI between the two experiments (Fig. 1). The ADT difference of <2 °C could
have altered the ﬂowering time by a few days, but probably would not affect the
process of ﬂoral evocation. However, the difference in DLl between the two
experiments was signiﬁcant (~5 mol'm'z'd"). Additionally, during the initial 4
weeks of forcing, when most plants made the transition from vegetative to
reproductive growth, the difference in DLl between the two experiments was
even larger (~13 mol‘m’z'd"). Based on these differences, we postulate that
following shorter durations of vemalization or SD treatment, a high DLI promoted
ﬂowering and/or a low DLI inhibited ﬂowering.

During both experiments, increasing duration of either inductive treatment
for up to 42 d decreased time to visible inﬂorescence and anthesis. The number
of inﬂorescences produced also saturated after 42 d of vemalization or SD
treatment in experiment I and after SD treatment in experiment ll. However, all
durations of vemalization induced similar number of inﬂorescences in experiment
ll. Due to significantly delayed ﬂowering, LD control plants developed additional
lateral shoots compared to vernalized plants in experiment I. In experiment ll,
delayed ﬂowering appeared to correlate with lateral shoot development, although
this was not evident in vernalized plants in experiment I. In experiment I, plants

had more lateral shoots following increasing durations of SD and this may have

130

 

 

 

been due to formation of additional laterals during SD treatments. However, due
to the large variability in the number of vegetative laterals produced following 20-
d SD treatment in experiment II, we can not conﬁrm this. In experiment I, the two
LD control plants ﬂowered on a lateral shoot rather than the terminal and hence,
both plants were signiﬁcantly shorter than the vernalized plants at anthesis.
Overall, the inductive treatments and their durations did not consistently affect

plant height at anthesis in both experiments.

Comparing Efﬁcacy of Vernalization Treatment With SD Treatment in
Regulating Flowering Responses. In experiment I, at each duration of inductive
treatment from 14 to 56 d, vemalization and SD treatment were equally effective
in promoting ﬂowering and elicited similar ﬂowering responses at anthesis except
number of lateral shoots (Fig. 2A-L). As previously explained, SD-treated plants
probably unfolded additional lateral shoots during the 20 °C treatment and hence
had more laterals at anthesis compared to plants exposed to 5 °C. In contrast, in
experiment ll, under the low DLl, short durations of vemalization treatment were
more effective in promoting ﬂowering compared to SD treatment. For example,
following 16 SD, no plants ﬂowered, whereas all plants vernalized for 16 d
ﬂowered. Hence, it appeared that under low DLI, vemalization treatment was
more effective in promoting ﬂowering than SD treatment based on ﬂowering
percentage. Once ﬂoral evocation occurred, ﬂowering time was similar for plants

receiving either inductive treatment. Interestingly, under the lower DLI, SD-

131

 

 

 

treated plants produced more lateral shoots but formed considerably fewer

inﬂorescences compared to vernalized plants.

Photoperiod During Vernalization. To determine if photoperiod during
vemalization affects the ﬂowering response of Coreopsis 'Sunray', plants were
vernalized at 5 °C under SD for 2 or 8 weeks and compared with plants

vernalized at 5 °C under a 16-h photoperiod in experiment I. The results indicate T7

 

that photoperiod during vemalization did not affect any ﬂowering response H

studied (data not presented). However, since the ﬂowering response was

 

saturated after 2-week inductive treatments based on ﬂowering percentage, we ' “'-
can not speculate if vemalization under SD would have been more effective

under shorter treatment durations.

Summary

Coreopsis 'Sunray' has an obligate requirement for vemalization or SD
treatment prior to exposure to ID for complete, rapid, synchronized and profuse
ﬂowering. The DLI during forcing may signiﬁcantly affect ﬂowering when the

inductive treatments are given for sub-optimal durations. Under the higher

 

forcing DLI, 14 d of SD or vemalization was sufﬁcient for complete ﬂowering

 

although longer treatment durations decreased ﬂowering time and increased the
number of inﬂrescences produced at anthesis. Overall, under the higher forcing
DLI, vemalization and SD treatments were similarly effective in affecting all

ﬂowering responses measured. Under the lower DLI, complete ﬂowering

132

occurred only following 216 d of vemalization or 42 d of SD treatment. Based on
ﬂowering percentage, short durations of vemalization treatment were more

effective than SD treatment in promoting ﬂowering under the lower forcing DLI.

 

 

 

 

133

Literature Cited

Damann, MP. and RE. Lyons. 1993. Juvenility, ﬂowering, and the effects of a
limited inductive photoperiod in Coreopsis grandiﬂora and C. Ianceolata J.

Amer. Soc. Hort. Sci. 118:513-518.

Ketellapper, H.J. and A. Barbaro.1966. The role of photoperiod, vemalization,
and gibberellic acid in ﬂoral induction, in Coreopsis grandiﬂora Nutt.
Phyton 23:33-41.

Yuan, M., W.H. Carlson, R.D. Heins and AC. Cameron. 1998. Effect of forcing
temperature on time to ﬂower of Coreopsis grandiﬂora, Gail/ardia
xgrandiﬂora, Leucanthemum xsuperbum, and Rudbeckia fulgida.
HortScience 33:663-667.

134

 

 

 

 

Figure 1. Average daily temperature (ADT; A, C) and daily light integral (DLI; B,
D) of Coreopsis grandiﬂora 'Sunray' plants forced at a 20 °C greenhouse
temperature setpoint and a 16-h photoperiod. Prior to forcing, plants were
provided inductive treatments for varying durations either by vernalizing at 5 °C
under 16-h photoperiod in 72-cell trays (A, B) or by growing under SD (9-h
photoperiod) at 20 °C in 13-cm square pots (C, D). Diamonds represent data
from experiment I and squares represent data from experiment ll. Each symbol
represents ADT or average DLI for an individual plant and was calculated from
transplant until anthesis and 105 d from transplant for ﬂowering and vegetative
plants, respectively. Error bars representing SE not presented since smaller than
the symbol size.

 

 

 

 

 

 

 

 

 

 

 

 

 

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135

Figure 2. The inﬂuence of vemalization treatment (AF) or SD treatment (G-L) on
ﬂowering percentage (A, G), time to visible inﬂorescence (B, H), time to anthesis
(C-l), number of inﬂorescences (D-J), number of lateral shoots (E, K) and plant
height (F, L) of Coreopsis grandiﬂora 'Sunray'. Plants were vernalized at 5 °C in
72-cell trays and exposed to short day (SD) treatment (9-h photoperiod at 20 °C)
in 13-cm square pots prior to forcing under a 16-h photoperiod at 20 °C.
Flowering percentage was computed 105 d after transplant. D-F and J-L were
counted on the day of anthesis. Diamonds represent data from experiment I and
squares represent data from experiment ll. MeaniSE of 9 and 10 replicates are
presented for experiment I and II, respectively.

136

 

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APPENDIX B:

DAILY LIGHT INTEGRAL AFFECTS THE CRITICAL SHORT-DAY AND LONG-

 

DAY PHOTOPERIODS OF THE SHORT-LONG-DAY PLANT COREOPSIS

éRANDIFLORA 'SUNRAY'

 

138 g

Daily Light Integral Affects the Critical Short-Day and Long-Day Photoperiods of

the Short-Iong-day Plant Coreopsis grandiﬂora 'Sunray'

Introduction
Coreopsis grandiﬂora ‘Sunray’ ﬂowers under a long-day (LD) photoperiod

following an inductive treatment of either vemalization or short-day (SD)

 

photoperiod (chapter IV; Appendix A). The critical photoperiods of Coreopsis

'Sunray' are unknown and therefore, the primary objectives of this study were to

 

determine the critical SD and LD photoperiod for complete, rapid, synchronized
and profuse ﬂowering of Coreopsis 'Sunray'. Some short-long-day plants
including Echinacea purpurea behave as intermediate photoperiod plants when
the constant photoperiod is shorter than the critical SD and exceeds the critical
LD photoperiod (Heide, 2004). Therefore, we also investigated the ﬂowering

responses of Coreopsis 'Sunray' under constant photoperiods.

Materials and Methods
Stockplant Management and Propagule Culture. In experiment I and II,

stockplants were developed and maintained as described in appendix A.

 

Cuttings were taken on 13 October 2004 and 18 February 2005 in experiments I
and II, respectively, and rooted for 2 weeks in 72-cell trays as described in
appendix A. Rooted plants were grown for 2 weeks in 72-cell trays in a glass

greenhouse under the same environmental and nutritional conditions as the

139

stockplants grown in the greenhouse. Subsequently, plants were transplanted in
13-cm square containers containing the same media used for the stockplants
and eight plants were placed under each photoperiod treatment combination in

the greenhouse.

Critical Photoperiod Treatments. To determine the critical SD photoperiod,
plants were placed under a 10-, 11-, 12-, 13- or 14-h photoperiod for 3 weeks
and then, forced under a 16-h photoperiod. To determine to critical LD
photoperiod, plants were placed under a 9-h photoperiod for 3 weeks and then
forced under a 12-, 13-, 14- or 15-h photoperiod. Additionally, plants were
placed under constant 11, 12, 13, 14 or 15-h photoperiod during the entire
course of the forcing.

The 9-h photoperiod was provided by a blackout system that opened from
0800 to 1700 HR. During the 9 h, plants received natural sunlight supplemented
by high-pressure sodium (HPS) lamps that turned on when ambient light was
below 200 pmol'm‘z's’1 photosynthetic photon ﬂux (PPF) and ceased when
ambient light exceeded 400 umol'm'z's'1 PPF. HPS lamps provided an
additional 150 pmol'm’z's'1 PPF. 10- to 15-h photoperiods were provided by a

blackout system open from 0800 to 1700 HR and by extending the photoperiod

after 1700 HR by incandescent lamps (at 1-3 pmol'm'z's“) for the desired duration.

For example, under the 10-h photoperiod, plants received a 9 h photoperiod by a

combination of sunlight and HPS lamps and an additional 1 h of light by

140

 

 

 

incandescent lamps. The 16-h photoperiod was provided by a combination of

sunlight and HPS lamps from 0600 to 2200 HR.

Plant Culture and Climate Control. During all photoperiod treatments and forcing,

 

plants were in the greenhouse with a 20 °C temperature setpoint. The
greenhouse climate was controlled by a climate control computer and air

temperature was measured on each greenhouse bench and PPF was measured F;

 

on 9-, 12- and 16-h photoperiod benches as described in appendix A. -~..

 

Data Collection. The number of nodes on each plant was counted prior to
initiation of inductive treatments and at the initiation of forcing in both ‘
experiments. On the day of ﬁrst visible inﬂorescence and anthesis of ﬁrst
inﬂorescence, dates were recorded and time to visible inﬂorescence and anthesis
was computed starting from the beginning of force At anthesis, number of
ﬂowering lateral shoots and inﬂorescences were counted. Plants not ﬂowering
105 d after the start of forcing were considered vegetative. Average daily
temperature (ADT) and average daily light integral (DLI) were computed from the
start of inductive treatments until anthesis and 105 d for ﬂowering and vegetative

plants, respectively and are presented in Fig. 1. The ADT and average DLI for

 

126 d from the start of inductive treatments for experiments I and II were 19.9

and 21.8 °C and 9.1 and 16.6 mol'm'z'd", respectively.

141

Results and Discussion

Cn'tical Short-Day Photoperiod. In experiment I and II, 88 and 100% of
plants ﬂowered following SD treatment at s11-h photoperiod, respectively (Fig.
2A). In experiment I (lower DLI), no plants ﬂowered following treatment at 212-h
photoperiod, but in experiment ll (higher DLI), ﬂowering percentage after
exposure to 12-, 13- and 14-h photoperiod was 50, 75 and 75, respectively. In
both experiments, following 10- and 11-h photoperiod treatments, ﬂowering time
and node number at anthesis were similar (Fig. ZB-D). In experiment ll,
ﬂowering of plants treated at 212-h photoperiod appeared to be slightly delayed
compared to plants treated at S11-h photoperiod. During experiment ll, ﬂowering
was hastened compared to experiment I, which can be explained by elevated
plant temperatures due to warmer ADT and higher DLI during experiment ll.
However, the difference in the node number at anthesis between the two
experiments suggests that plants ﬂowered at an earlier developmental stage
under a high DLI. In both experiments, 10- and 11-h photoperiod treatments
elicited similar number of ﬂowering lateral shoots and number of inﬂorescences
(Fig. 2E and F). In experiment ll, S12-h photoperiod treatment resulted in more
ﬂowering lateral shoots and inﬂorescences compared to 211-h treatment.

Based on above data, the critical SD photoperiod for complete, rapid and
synchronized ﬂowering was 11 h. We postulate that the observed ﬂowering
following 212-h photoperiod in experiment ll could be attributed to the higher DLI
during experiment ll. Since an unsaturating 3-week duration was used for

inductive SD treatments, the ﬂowering responses were highly sensitive to forcing

142

 

 

 

 

DLI (appendix A). Further studies are necessary to determine the effect of
forcing DLI on the critical SD photoperiod following unsaturating and saturating
durations of SD treatments.

Cn'tical Long-Day Photoperiod. In experiment I, 100, 75 and 88% plants
previously treated with SD at 9-h photoperiod ﬂowered when forced under 513,
14 and 15-h photoperiods, respectively (Fig. ZG). All SD-treated plants ﬂowered
when forced under 212-h photoperiod in experiment ll. Based on time to visible
inﬂorescence and anthesis and number of nodes at anthesis, plants ﬂowered at a
similar time when forced under 213-h photoperiod in both experiments and

ﬂowering was delayed under the 12-h photoperiod (Fig. 2H-J). Plants ﬂowered

 

sooner in experiment ll compared to experiment I, and this could have been due
to elevated plant temperatures. However, the decreased number of nodes at
anthesis in experiment ll indicated that ﬂoral evocation was promoted under the
higher DLI. When forced under a 12-h photoperiod, plants produced fewer
ﬂowering lateral shoots compared to plants forced under 213-h photoperiod in
both experiments (Fig 2K). In both experiments, plants under a 12-h forcing
photoperiod had fewer inﬂorescences compared to plants under 213-h
photoperiod, and plants under 13-h photoperiod had fewer inﬂorescences

compared to plants under 214-h photoperiod (Fig. 2L). Plants had similar

 

number of inﬂorescences when forced under a 14- and 15-h photoperiOd in both
experiments.
Based on ﬂowering percentage, the critical LD photoperiod of Coreopsis

'Sunray' was 12 h; based on ﬂowering time and number of ﬂowering lateral

143

 

shoots, the critical LD photoperiod was 13 h; and based on the number of
inﬂorescences at anthesis, the critical LD photoperiod was 14 h. Our data also
indicate that DLI only slightly affected the ﬂowering responses at different critical

LD photoperiods.

Constant Photoperiods. In experiment I, no plants ﬂowered under constant
photoperiods of 11 to 15 h (Fig. 2M), whereas in experiment ll, ﬂowering
percentage was 0, 50, 88, 88 and 13 under 11-, 12-, 13-, 14- and 15-h
photoperiod, respectively. Thus, ﬂowering under constant photoperiods was
incomplete and inconsistent and it is likely that ﬂowering reported in experiment II
was inﬂuenced by the DLI. Although average time to visible inﬂorescence and
anthesis and number of nodes at anthesis indicate that ﬂowering was hastened
as forcing photoperiod increased from 11 to 15 h, due to incomplete ﬂowering at
all constant photoperiods, no conclusions can be made on the effect of constant
photoperiods on ﬂowering time (Fig. 2N-P). Similarly, due to incomplete
ﬂowering, no conclusions can be made on the effect of constant photoperiod on
number of ﬂowering shoots and number of inﬂorescences at anthesis (Fig. 20

and R).

Summary
Based on the results from both experiments, the critical length of SD and
LD photoperiods should be 11 and 12 h, respectively, for complete ﬂowering of

Coreopsis 'Sunray'. Increasing the LD photoperiod from 12 h to .214 h decreased

144

 

 

 

 

ﬂowering time and increased the number of ﬂowering shoots and number of
inflorescences at anthesis. Incomplete and inconsistent ﬂowering occurred at
constant photoperiods. Overall, DLI affected ﬂowering responses under all

photoperiod treatment combinations tested.

145

 

 

 

Literature Cited

Heide, OM. 2004. Dual induction rather than intermediate daylength response of
ﬂowering in Echinacea purpurea. Physiol. Plant. 120:298-302.

 

 

 

 

146

Figure 1. Average daily temperature (ADT; A) and average daily light integral
(DLI; B) of Coreopsis grandiﬂora 'Sunray' plants grown in a 20 °C greenhouse.
ADT and average DLI were computed from the beginning of the inductive
treatments until anthesis and 105 d from initiation of forcing for ﬂowering and
vegetative plants, respectively. Dark lines represent the data from experiment I
and grey lines represent data from experiment ll.

 

26 ...A.

24 up...

22 ...... ._

ADT (°C)

:2!
20 , .

 

 

 

 

3o

20 ..

 

DLI (mol'm'z'd'1)

 

 

 

Day of the experiment

147

 

 

 

Figure 2. Flowering responses of Coreopsis grandiﬂora 'Sunray' plants following
critical SD photoperiod of 10 to 14 h (A-F), critical LD photoperiod of 12 to 15 h
(G-L) and constant photoperiod of 11 to 15 h (H-R). Following critical SD
treatment for 3 weeks, all plants were forced under a 16-h photoperiod and prior
to critical LD treatment, all plants were treated with a 9—h photoperiod for 3
weeks. The inﬂuence of photoperiod treatments on ﬂowering of Coreopsis
grandiﬂora 'Sunray' was assessed based on ﬂowering percentage (A, G, M), time
to visible inﬂorescence (B, H, N), time to anthesis (C, l, 0), number of nodes at
anthesis (D, J, P), number of ﬂowering lateral shoots (E, K, O) and number of
inﬂorescences (F, L, R). Flowering percentage was computed 105 d after
initiation of force. D-F, J-L and P-R were counted on the day of anthesis.
Diamonds represent data from experiment I and squares represent data from
experiment ll. MeantSE of 8 replicates are presented.

148

>wing
5 h

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lime

ll

 

Percent ﬂowering

Time to
visible bud (d)

Number of Time to
lateral shoots nodes at anthesis anthesis (d)

Number of ﬂowering

Number of
buds and ﬂowers

80 ..
60 .
40«

20

80 >
60 ‘
40 0
20 r

24‘

21

18 ..

15‘

Critical SD

Critical LD

Constant photoperiod

 

 

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149

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