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ASEXUAL PROPAGATION OF ANEMONE HUPEHENSIS
AND ANEMONE XHYBRIDA BY A
ROOT-PLUG METHOD

presented by

JOAQUIN ANDRES CHONG

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6/01 chlRC/DaloDuepGS—p. 15

ASEXUAL PROPAGATION OF ANEMONE HUPEHENSIS
AND ANEMONE XHYBRIDA BY A
ROOT-PLUG METHOD
By

Joaquln Andrés Chong

A THESIS

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

MASTERS OF SCIENCE

Department of Horticulture

2000

ABSTRACT

ASEXUAL PROPAGATION OF ANEMONE HUPEHENSIS
AND ANEMONE XHYBRIDA BY A
ROOT-PLUG METHOD

By
Joaquin Andres Chong

Anemones are commonly propagated by root cuttings; however, there is
general uncertainty and unfamiliarity with this procedure. Studies were
conducted to determine the inﬂuence of seven photoperiods and two cold
treatments on the dry weight partitioning of anemone and subsequent impact on
root cutting development. Shoot and root dry weights were obtained after 16.5-
or 15-weeks of growth at 20 °C for Anemone hupehensis and A. xhybn'da
respectively, following either 0- or 15-weeks at 5 °C. Time to visible ﬂower bud
and to visible root bud, number of root-buds, and plant heights were determined
after treatments for A. xhybn'da. Root dry-weight partitioning decreased with
increasing photoperiod and following cold treatment; however, cold increased
root-bud number under photoperiods 5 12 h. A maximum of 60% and a minimum
of 32% Of dry weight were allocated to roots under 10-h photoperiods without
cold, or 24-h photoperiods with cold, respectively.

A study was conducted to test a new method for more efﬁcient
propagation by root cuttings. A root-plug (RP) technique was developed that
consisted of growing mother plants in a 1.5 L container with internally divided
compartments, specifically a 128- and a 288-cell plug tray stacked vertically or
three 288-cell plug trays horizontally or vertically oriented. Neither plant age
(7.5- or 28-week) nor cold treatment (0- or 15-week) affected root plug
regeneration. Top trays produced the most RPs, up to an average of 17.3.
Root-plug production in plug trays decreased with increasing distance from the
crown. Results with vertically oriented trays suggested that roots had to elongate

to thicken, as middle trays produced more RPs than side trays.

In the memory of my father Victor Chong Kam
because he always wanted us to study

and to my family Pilar, Maxim,

Victor, and Viviana who

I deeply love.

ACKNOWLEDGEMENTS

I would like to thank my major professor Dr. Royal Heins for accepting and
giving me the opportunity to achieve new goals in life and work as his graduate
student and research assistant. I would also like to thank the members of my
guidance committee: Dr. Art Cameron and Dr. William Carlson for their guidance
over these years, and Dr. Kenneth Poff for providing thoughtful knowledge and
the opportunity to visit MSU as a summer intern.

It has been a pleasure be part of the best ﬂoriculture group. I would like to
express a deep gratitude to my friend and coworker Erik Runkle for his support
and all the questions he answered over these last two years, thanks for providing
knowledge to my life.

I must also thank Emily Clough, Beth Fausey, Alison Frane, Hongwen
Gao, Taskahiro Hayashi, Kathy Kelly, Hyeong Hey Kim, Bin Liu, Mary-Slade
Morrison, Genhua Niu, Shi-Ying Wang, and Cathy Whitman for all the work and
fun we had together. Special thanks to Dan Tschirhart, David Joeright and Ron
Wik whose assistance and support were crucial for the completion of this

research.

TABLE OF CONTENTS

LIST OF TABLES ............................................................................................... Vll
LIST OF FIGURES ............................................................................................ VII
SECTION I
LITERATURE REVIEW ........................................................................................ 1
Plant Background .............................................................................................. 2
Plant Description ............................................................................................ 4
Plant Production ............................................................................................ 5
Root-Cutting Propagation .................................................................................. 5
Advantages of Root-cutting Propagation ....................................................... 9
Disadvantages of Root-cutting Propagation .................................................. 9
Factors affecting root-buds .............................................................................. 10
Secondary Thickening ................................................................................. 10
Seasonal Fluctuations ................................................................................. 13
Hormonal control ......................................................................................... 16
Conclusion ...................................................................................................... 1 8
References ...................................................................................................... 20
SECTION II

EFFECTS OF PHOTOPERIOD AND COLD TREATMENT ON DRY WEIGHT
PARTITIONING OF ANEMONE HUPEHENSIS AND ANEMONE XHYBRIDA,
AND A ROOT-PLUG METHOD FOR PROPAGATION OF ROOT CUTTINGS. .24

Abstract ........................................................................................................... 26
Introduction ..................................................................................................... 27
Materials and methods .................................................................................... 31
Plant material ............................................................................................... 31
Plant culture ................................................................................................. 32
Dry weight partitioning experiment ............................................................... 32
Root-plug experiment .................................................................................. 33
Experimental settings .................................................................................. 35
Data collection ............................................................................................. 36
Results ............................................................................................................ 37
Dry weight partitioning experiment ............................................................... 37
Root-plug experiment .................................................................................. 39
Discussion ....................................................................................................... 40
Dry weight partitioning experiment ............................................................... 4O
Root-plug experiment .................................................................................. 42
References ...................................................................................................... 44
APPENDIX

EFFECTS OF PHOTOPERIOD TRANSFERS AND COLD TREATMENTS ON
THREE ASTERACEAE SPECIES, COREOPSIS GRAND/FLORA “EARLY
SUNRISE’, LEUCANTHEMUM XSUPERBUM ‘SNOW LADY’ AND RUDBECKIA
FULGIDA ‘GOLDSTURM’. .................................................................................. 52

Abstract ........................................................................................................... 54

Introduction ..................................................................................................... 55
Materials and methods .................................................................................... 59
Plant material ............................................................................................... 59
Initial Photoperiod Treatments ..................................................................... 59
Photoperiod transfers and cold treatments .................................................. 60
Plant culture ................................................................................................. 62
Temperature Settings .................................................................................. 62
Data Collection ............................................................................................ 63
Results ............................................................................................................ 65
Dual-photoperiod experiment ...................................................................... 65
Cold treatment experiment .......................................................................... 66
Discussion ....................................................................................................... 69
References ...................................................................................................... 73

vi

LIST OF TABLES

SECTION I

Table 1. Anemone classiﬁcation by ﬂower timing and root morphology ................ 2

Table 2. Hudson’s (1955) approach to classiﬁcation of modes of regeneration
based on the study of relations between parent plants and root suckers ...... 7

LIST OF FIGURES
SECTION I
Figure 1. Fibrous roots similar to summer-autumn anemones’ roots (Graf, 1992)
....................................................................................................................... 3
Figure 2. Types of regeneration from roots (Hudson, 1956) ................................ 8

Figure 3. Flow of photosynthates (PS; Forrester, 1968; Wilson, 1975;). Boxes
are levels, or amounts, valve symbols are rates of ﬂow, and the oval is an
external source or sink. ................................................................................ 11

Figure 4. Juvenility Triangle. The closer to the crown the best possible
regeneration (Dir and Heuser, 1987) ........................................................... 12

Figure 5. Hudson’s (1954) description of seasonal cycles of Rubus idaeus L.
root cuttings ................................................................................................. 13

SECTION II

Figure 1. Drawing of root-plug tray treatments for Anemone hupehensis (A and
B) and A. xhybn'da (A, B and C) root-plug experiment. Tray treatments
128/288, 288/288/288, and 288-288/288 are represented in ﬁgures A, B, and
C, respectively. ............................................................................................ 46

Figure 2. A: The effects of photoperiod on Anemone hupehensis root (unﬁlled
circles) and shoot (ﬁlled circles) dry weights and visible ﬂower-bud
percentage (unﬁlled diamonds). B: Dry root to shoot ratios in response to
photoperiod. Error bars are 95% conﬁdence intervals and for clarity are
offset to the right of data points for shoot dry weights. L = linear; Q =
quadratic trends. ”5'" Nonsigniﬁcant or signiﬁcant at P 5 0.01. .................. 47

Figure 3. The effects of photoperiod on the growth of Anemone xhybn'da
‘Whirlwind’. A: Dry weight of uncooled plants and visible ﬂower bud
percentage measured at 15 weeks. B: Dry weight of plants cooled for 15
weeks and visible ﬂower bud percentage. In A and B ﬁlled circles represent
shoot dry weight; unﬁlled circles root dry weight. C: Unﬁlled squares
represent root : shoot ratios of uncooled plants; ﬁlled squares, root : shoot
ratios of plants cooled 15 weeks. Error bars are 95% conﬁdence intervals
and for clarity are offset to the right. L = linear; Q = quadratic trends. Ns'*'***
Nonsigniﬁcant at P 5 0.05 or 0.001 .............................................................. 48

vii

Figure 4. The effects of photoperiod on days to visible root bud (A), root bud
number (B), and canopy height (C) of Anemone xhybn'da ‘Whirlwind’.
Unﬁlled symbols represent uncooled plants grown for 15 weeks. Filled
symbols represent plants after 15 weeks of 5 °C and 15 weeks’ growth.
Error bars are 95% conﬁdence intervals and for clarity are offset to the right
of data points for 5 °C treated plants. L = linear; Q = quadratic trends. Ns'*'***
Nonsigniﬁcant or signiﬁcant at P 5 0.05 or 0.001, respectively. ................... 49

Figure 5. The regeneration of Anemone xhybida cut after 15 weeks of growth
and measured after 5 weeks of root-bud regeneration. Total root plugs
produced from 128l288 (A) and 288/288/288 (B) tray treatments. Root plugs
produced per level from 128l288 (C) and 288/288/288 (D) tray treatments.
Error bars are 95% conﬁdence intervals. Unﬁlled circles represent raw data
and for clarity are offset to the right when the same value is repeated. ....... 50

Figure 6. The regeneration of Anemone hupehensis out after 16.5 weeks of
growth and measured at 5 weeks of root-bud regeneration. Total root plugs
produced from 128l288 (A), 288/288/288 (B), and 288-288/288 (C) tray
treatments. Root-plugs produced per level from 128l288 (D), 288/288/288
(E), and 288-288/288 (F) tray treatments. Error bars are 95% conﬁdence
intervals. Unﬁlled circles represent raw data and for clarity are offset to the
right when the same value is repeated. ....................................................... 51

APPENDIX

Figure 1. Schematic representation of photoperiod transfer experiment. Black
ﬁlled bars represent initial photoperiod under 9- (SD) or 16-h (LD)
photoperiods and gray bars represent the transfer photoperiod duration.
Plants were under the initial photoperiod for 10, 11, or 13 weeks for
Coreopsis, Leucanthemum, and Rudbeckia, respectively. After the initial
photoperiod treatment, plants were moved to the opposite photoperiod for 0,
1, 2, 3, 4, 5, 6, 8, or 10 weeks and then transferred back to the initial
photoperiod if anthesis was not reached. .................................................... 74

Figure 2. The effects of LD-SD-LD (A ; SD = short days; LD = long days) and
SD-LD-SD (B) on days to visible bud, days to ﬂower, ﬂowering percentage,
ﬂower bud number (C), and plant height (D) of Coreopsis grandiﬂora ‘Early
Sunrise’. Unﬁlled circles represent LD-SD-LD (NIP) treatment and black-
ﬁlled circles represent SD-LD—SD (LIP) treatments in (C) and (D). Error bars
are 95% conﬁdence intervals. L = linear; Q = quadratic trends. NS,**,***
nonsigniﬁcant or signiﬁcant ot P 5 0.01 or 0.001, respectively. ................... 75

Figure 3. The effects of SD-LD-SD (SD = short days; L0 = long days) on days to

visible bud, days to ﬂower, ﬂowering percentage (A), plant height (B), and
ﬂower bud number (C) of Leucanthemum xsuperbum ‘Snow Lady’. L =

viii

linear; Q = quadratic trends. NS,*,*** nonsigniﬁcant or signiﬁcant at P 5 0.05
or 0.001, respectively ................................................................................... 76

Figure 4. The effects of LD-SD-LD (A; SD = short days; L0 = long days) and SD-
LD-SD (B) on days to visible bud, days to ﬂower, ﬂowering percentage,
ﬂower bud number (C), and plant height (D) of Rudbeckia fulgida
‘Goldsturm’. Unﬁlled circles represent LD-SD-LD (NIP) treatment and black-
ﬁlled circles represent SD-LD-SD (LIP) treatments in (C) and (D). Error bars
are 95% conﬁdence intervals. L = linear; Q = quadratic trends. NS,*,***
nonsigniﬁcant or signiﬁcant at P 5 0.05 or 0.001, respectively. ................... 77

Figure 5. The effects of cold treatment on days to ﬂower (A), plant height (B),
and ﬂower bud number (C) of Coreopsis grandiﬂora “Early Sunrise”. Days to
ﬂower were calculated from seeding and the end of cold. Black-ﬁlled
symbols represent SD before cold; unﬁlled symbols, LD before cold. Error
bars are 95% conﬁdence intervals. L = linear; Q = quadratic trends. NS,*,***
nonsigniﬁcant or signiﬁcant at P 5 0.05 or 0.001, respectively. ................... 78

Figure 6. The effects of cold treatment on days to ﬂower (A), plant height (B) and
ﬂower bud number (C) of Leucanthemum xsuperbum ‘Snow Lady’. Days to
ﬂower were calculated from seeding and the end of cold. Black-ﬁlled
symbols represent SD before cold. Error bars are 95% conﬁdence intervals.
L = linear; Q = quadratic trends. NS,*,**,*** nonsigniﬁcant or signiﬁcant at P
5 0.05, 0.01, or 0.001, respectively. ............................................................. 79

Figure 7. The effects Of cold on days to ﬂower (A), lateral ﬂowering percentage
(B), ﬂower bud number (C), and plant height (D) of Rudbeckia fulgida
‘Goldsturm’. Days to ﬂower were calculated from seeding and the end of
cold. Black-ﬁlled symbols represent SD before cold; unﬁlled symbols, LD
before cold. Error bars are 95% conﬁdence intervals. L = linear; Q =
quadratic trends. NS,*,**,*** nonsigniﬁcant or signiﬁcant at P 5 0.05, 0.01, or
0.001, respectively. ...................................................................................... 80

SECTION I
LITERATURE REVIEW

Plant Background

Anemones are members of Ranunculaceae and are commonly known as
Windﬂower or Lily-Of-the-ﬁeld. Ranunculaceae contain more than 120 species
distributed from zones 4 to 10. The minimum temperature range they tolerate is
—35 to 5°C (Bailey, 1978).

Anemones can be divided into three main groups according to ﬂowering

season and their root morphology (Table 1).

Table 1. Anemone classiﬁcation by ﬂower timing and root morphology

 

:4ng Spring Early summer Summer/autumn
Root Morphology Tuberous rhizomes Tuberous roots Fibrous roots

 

Spring-ﬂowering anemones are characterized by subterranean knobby,
tuberous, bulblike rhizomes capable of producing new shoots. These anemones,
such as A. apennina L., A. blanda Schott 8. Kotschy, A. canadensis L., A.
demissa Hook F ._ & T. Thoms., A. xlessen', A. nemorosa L., and A. sylvestn's L.,
are hardy from USDA zones 4 to 9, which have minimum temperatures from —35
to —1°C. USDA climate zones are based on the average annual minimum air
temperature.

Early-summer-ﬂowering anemones have enlarged, ﬂeshy, subterranean
tuberous roots; species include A. coronan’a L., A. x fulgens (DC.) Rchb. and A.
pavonina Lam. Early summer anemones are fully hardy from USDA zones 4 to
10, which have minimum temperatures of —35 to 5°C.

Summer-autumn-ﬂowering anemones are often called Japanese

anemones and have ﬁbrous roots. They are important perennial species used

as cut ﬂowers and potted plants. In mid Michigan, these anemones ﬂower from
mid-August to early October. They are slow-growing perennials that can be
difﬁcult to establish and require well-drained and aerated soil, especially when

ﬁrst transplanted (Armitage, 1997).

Summer-autumn anemones have a
determinate growth habit: the shoot
growth stops with ﬂowering. Leaves and
shoots die back after ﬂowering and
decompose during the winter, leaving

roots with root buds that ovenNinter.

 

Anemones from this class are fully hardy

Figure 1. Fibrous roots similar _ _
to summer-autumn anemones' In USDA zones 5 to 9, which have

roots (Graf, 1992) minimum temperatures from —23 to 1° C.
Species include A. hupehensis var. japonica (Thunb.) Bowels & Stearn, A.
xhybrida Paxt., A. japonica, A. tomentosa (Maxim) P'ei., and A. vitifolia Buch.
Ham. ex DC. (Jelitto and Schacht, 1995; Armitage, 1997).

The most common anemones of the summer-autumn ﬂowering group are
A. hupehensis var. japonica and A. xhybn'da. Anemone hupehensis var. japonica
is from China (1844) although abundant in Japan, and is 60- to 75-cm tall, with
ﬁve to seven rosy mauve sepals; some plants have male-sterile ﬂowers.
Anemone xhybn'da ﬂowers about a week later than A. hupehensis var. japonica

and is 40-cm higher and 14-cm wider. Many A. xhybn'da cultivars have semi- or

double ﬂowers. Anemone xhybn'da ‘Whirlwlnd' was introduced in 1887 and has

semidouble white ﬂowers (Graham, 1990). Anemone xhybrida is a cross
between A. hupehensis var. japonica and A. vitifolia (Bailey, 1978).

Plant Description

Anemone plants are composed of leaves and petioles, which form the
majority of the upper plant structure. The main stem is nearly covered by the leaf
stipules during the nonreproductive stage. As the plant enters its reproductive
cycle, the main stem elongates producing ﬂower shoots that carry non-base
leaves and ﬂower buds. Petioles, shoots, and leaves are pubescent. Leaves are
serrated and, as with Hedera helix, change morphology as the plant matures
(Miller and Goodin, 1976). Juvenile anemones initially produce single-lobed
leaves; as the plant matures, there is a gradual change in subsequent leaves to a
trilobed (mature) form. The ﬁnal mature leaf form is a compound trilobed leaf
(Chong et al., 2000).

Plant morphological characteristics change as plants develop (Whyte,
1938). As with many plants (O’Rourke, 1951), anemones reproduced from root
cuttings undergo a reversion to juvenility, or return to a juvenile phase. The
reversion has been characterized in some plants by morphological changes such
as leaf shape, arrangement on the stem, leaf retention on deciduous plants,
thominess, pubescence, and more easily rooted tissues (O‘Rourke, 1951; Fisher,
1961 ). In some cases, reversion to juvenility is false or transitory. Juvenile apple
root cuttings taken from root sprouts ﬂower signiﬁcantly faster than an apple plant

produced from seed. Early ﬂowering suggests a putative juvenile stage and a

possible separation of the juvenile characteristic of easy rooting from a mature
characteristic of ﬂowering (Robinson and Schwabe, 1977a).

Plant Production

Anemones are propagated by seed, root cuttings, and division (Perry,
1998). Seed-propagated anemones are variable and not true to type. Arrnitage
(1997) describes root-cutting propagation as more effective than shoot-division
propagation. Root cuttings are used as the main source of commercially
propagated material. Root buds will form from thick roots that are approximately
7-to 10-cm long and placed vertically in a moist, well-drained medium (Armitage,
1997)

Fisher (1961) described a method for root-cutting propagation of anemone
and other perennials. Mature, one-year-old anemone plants are dug and
harvested during mid-November. Root cuttings are placed in grape crates with
soil and stored in a cold greenhouse until root buds emerge and roots grow.
Then plants are planted in pots for sale in late spring or are planted in the ﬁeld.
Root-Cutting Propagation

Roots perform different functions, including stabilization and water and
mineral absorption. They also provide propagation material such as root
cuttings, sprouts, and tubers. Some roots help certain plants survive adverse
conditions by producing root buds (Peterson, 1975). Quaking aspen trees for
example, are found from Arctic regions to Mexico and survive ﬁres and icing
conditions successfully because of their high root-budding capacity estimated to

be one million sprouts per acre (Madison, 1999).

Root-cutting propagation depends on the ability of certain roots to produce
root buds, which are deﬁned as subterranean adventitious shoot buds located on
lateral roots (Horvath, 1998). In some plants, root buds are responsible for the
production of “root sprouts” or “root suckers." Root suckers emerge from
subterranean roots and should not be confused with stem suckers that emerge
from subterranean stems (Hudson, 1956). Root suckers emerging from a deep
root push through the soil, etiolate, and Often have a greater potential for rooting,
even in species that do not normally root from stem tissue (Hudson, 1956).
Hudson called the subterranean part of the sucker the “sucker base” and roots
emerging from it “stem roots.”

Macdonald (1986) described root cutting as a technique in which roots
capable of developing root buds (shoots, suckers) are cut into individual pieces
capable of regenerating plants. These root sprouts can be used as stem cuttings
because they undergo a rejuvenility process, which makes them easy to root
(O’Rourke, 1951 ). Stem cutting of root sprouts is not possible in Anemone.

Several authors (Wobst, 1868; Lindsay, 1877; anonymous, 1882; Fisher,
1961; Stoutemyer, 1968; Flemer, 1983; Macdonald, 1986; Bath and Jones, 1994;
Fossel, 1998; Sanchez, 1999) have listed root bud—producing plants. Wobst
(1868) found that the following families contain species that can reproduce by
root cuttings: Apocynaceae, Asclepiadaceae, Bignoniaceae, Campanulaceae,
Geraniaceae, Leguminosae, Papaveraceae, Passiﬂoraceae, Plumbaginaceae,

Rosaceae and Rubiaceae.

Root buds can be classiﬁed according to their mode of origin as

“additional” or “reparative.” Additional root buds emerge from undisturbed roots,

and reparative root buds emerge
from injured or senescent roots
(Bosela, 1997). However, this
classiﬁcation system is

subjective, since all roots
encounter stresses and have
minor injuries during their normal
growth and development. In
some species, these stresses or
injuries might be enough to
cause root bud formation
(Bosela, 1999). Horticulturally,
Hudson (1956) classiﬁed root
buds by their position as either
lateral buds that grow on the
side of roots or terminal buds
that grown on ends of roots that
are injured or cut off. Hudson

(1955b) proposed a classiﬁcation

 

Table 2. Hudson’s (1955b) approach to
classiﬁcation of modes of regeneration
based on the study of relations between
parent plants and root suckers

 

Type

Description

 

A

Root suckers growing near the
parent plant, to which they
normally remain attached.

 

Suckers produced naturally
from uniniured roots, where
the connecting root ultimately
dies.

 

Suckers rarely produced
except in response to root
iniurv. though without the
root’s being wholly severed.

 

New plants produced from
pieces of root severed from
the parent but remaining in
situ, provided the assimilating
root system is not disturbed.

 

Regeneration possible from
pieces of root, severed at both
ends and with no undisturbed
roots (the “root cutting” of
horticulture).

 

Roots capable of regeneration
after only special treatment
(e.g., grafted with a nurse
scion).

 

 

 

Plants apparently unable to
produce root suckers under
any circumstances.

 

 

 

 

 

by mode of plant regeneration based on relations between parts of the parent
and the new plant. Root cuttings were classiﬁed into seven different types
(Table 2).

Regeneration from root buds of types B and C occurs with a connecting
root between parent shoot and root bud. The root bud receives continual
supplies of water and polysaccharides from established roots and parent shoots,

which fosters regeneration. In type D, connecting roots between parental shoot

are eliminated, while in type E, root ®
buds are not connected with either
aerial shoot or assimilating root -

A 3 C

systems. Root cuttings (type E)

depend on their own resources.

 

Plants of types E and B can be

propagated by TOOt CUtIIIIQS. DUI Figure 2. Types of regeneration from

roots (Hudson, 1956)
those of types C and D must be

propagated in situ (Hudson, 1955b 1956).

Hudson (1955b) found that the response of a piece of root depends on
two factors: the condition of the parent plant, which determines the capacity of
the cutting to regenerate, and the effect of the environment and orientation of the
cutting, which determine its performance. Plant capacity refers to required
factors for regeneration success before removal of root cuttings from the mother
plant. Plant performance refers to the environmental and hormonal factors that

affect regeneration after root cuttings are separated from the mother plant. Root-

cutting success depends on the performance of the cutting and the capacity of
the mother plant.

Advantages of Root-cutting Propagation

Root-cutting propagation is advantageous for plants that can produce root
buds and are difﬁcult to root from stems, do not bear seed, or do not produce
seed that is true to type (Fisher, 1961; Macdonald, 1986). Root cuttings are
easier to acclimatize than shoot cuttings (Hudson, 1955b). Root cuttings can
eliminate the need for rootstocks, budding, and grafting (Robinson and Schwabe,
1977a). Root cuttings might have a longer life than grafted clones because
grafting might cause scion-under—stock incompatibilities (Flemer, 1983). In
general it is cheaper to propagate a root cutting than to grow an understock and
then graft or bud it (Flemer, 1983).

Disadvantages of Root-cutting Propagation

There are several disadvantages to root-cutting propagation. Root
cuttings have been described as one of the least frequently used and least
desirable methods of vegetative propagation (Fisher, 1961; Heuser, 1977;
Flemer, 1983). There are several reasons for this undesirability, among them
that root-cutting propagation is a tedious and messy procedure. Root cuttings
are more difﬁcult to gather than aerial cuttings (Flemer, 1983). The method is
labor intensive, the plants must be dug or the soil excavated to expose the roots,
and the procedure must be carried out during the fall or winter (Heuser, 1977).
There might be year-to-year differences in root material. In some years it might

be difﬁcult to obtain large quantities of root-cutting material (Flemer, 1983; Ede et

al., 1997). Chimeric material will most likely not come true to type if the mutated
section is not propagated or if the mutation is in the shoot section. Little research
has been aimed at the quantiﬁcation of root cuttings per plant (Macdonald, 1986).
Without knowledge of seasonal effects on the roots, it is impossible to
morphologically distinguish roots capable of producing root buds (Hudson, 1954).
Finally, there is a general uncertainty about and unfamiliarity with this procedure,
making it unattractive.
Factors affecting root-buds

Several internal and environmental factors affect root bud initiation and
development in root bud—producing plants: secondary thickening, seasonal
ﬂuctuations (cold and photoperiod), and hormonal control (Hudson, 1954;
Robinson and Schwabe, 1977b).

Secondary Thickening

Hudson (1955a) described bud—producing roots as any piece (2 to 3 cm)
of secondary thickened root capable of growing and developing during a certain
season. Roots have to mature before they are capable of root-bud production. A
mature root has a primary cortex and has developed some secondary tissue.
“Ripeness to regenerate” is the developmental stage a root has to reach before
root buds can regenerate (Dore, 1955). Wilson (1975) described the distribution
of secondary thickening in tree root systems.

Photosynthates (PS) are transported from the chloroplastic tissue to the
entire plant (Figure 3). Some PS are trapped in sink tissues and others ﬂow to

roots, causing secondary root thickening. The ﬂow of PS to the roots is regulated

1O

Figure 3. Flow of photosynthates (PS; Forrester, 1968; Wilson, 1975;). Boxes
are levels, or amounts, valve symbols are rates of ﬂow, and the oval is an
external source or sink.

PS STORED

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

TRANSPORT STORAGE
\/ PS IN V PS IN
:ﬁLHOEM AVAILABLE SECONDARY
11 POOL G) THICKENING
Z O
‘2 E
k I

 

 

 

 

 

 

 

 

I> RESPIRATION

 

 

 

 

 

PS RESPIRED

 

by rate of transport, storage, respiration, growth, and hormone interaction
(Wilson, 1975). Factors that affect shoot growth affect root growth, and vice
versa. Plants whose growth and photosynthesis is slow because of stress will
have reduced root growth because less PS and hormones are transported to the
roots (leon, 1975). In potato, for example, short-day photoperiods promote
tuberization and long-day photoperiods delay it. However, the long-run yield of
potato is better under long days than under short days, probably because less
PS are produced during short days due to less light availability (Gregory, 1965).
Generally in order to obtain thick roots, plants must have healthy photosynthate-

producing shoots and leaves.

11

As previously mentioned, root buds develop only from secondary

thickened

thin feeding roots. Root-bud

proliferation and survival increase as

root diameter and length increase
presumably because available
carbohydrate reserves are greater.

Thick and longer roots allow numerous,
vigorous, longer root sprouts (Robinson
and Schwabe, 1977a; Lawes and Sim,

1980). Longer thicker root cuttings

roots, not from short-lived

 

 

N ,’ , Cone of
/ \ juvenility
Maximum If 3‘
juvenility ,v -I‘\\
/T ,1 .1; Q I‘
( I v
>:;.
Maximum
juvenility
Figure 4. Juvenility cone. The

closer to the crown, the better the
possibility of regeneration (Dir and
Heuser. 1987).

produce better root sprouts than shorter roots (Robinson and Schwabe, 1977a;

Ghani and Cahalan, 1991).

The PS transport capacity increases as root

circumference increases (Wilson, 1975). However, root thickness and sprouting

capabilities might be confounded with positional effects (McMillan, 1980).

Gradients in hormone concentration change within roots as root position gets

farther away from the crown (Figure 4).

increasing distance from the crown.

Also, root diameter decreases with

Studies on Paulownia spp. demonstrated that regenerative ability of root

cuttings is highly correlated with root diameter and is not diminished with

increasing distance (horizontally or vertically) from the root crown (Ede et al.,

1997)

12

Seasonal Fluctuations

More than a hundred years ago, Wobst (1868) suggested that better
regeneration from root cuttings occurred when cuttings were taken in early spring
before the mother plant developed. He also described a method for nonhardy
plants, which consisted in extracting roots in autumn and overwintering them in
sand or soil in a frost-free environment, then cutting and growing them in the
spring. An understanding of the mechanisms behind this empirical knowledge
did not start until the mid-19505, when Hudson published several scientiﬁc
papers about the effects of seasonal ﬂuctuations on root regeneration, growth,
and development. Different physiological plant mechanisms are stressed as
temperature and photoperiod change with the seasons.

Working with raspberry root cuttings (Rubus idaeus L.), Hudson (1954)
found a strong seasonal response to root-bud regeneration. Root cuttings taken
at certain periods of the year regenerated better than others did. He classiﬁed

these months of good regeneration as the “on” season (Figure 5).

 

 

 

 

August September October November December January February March April May June July
on off

 

 

 

 

 

 

intermediate intermediate

 

Figure 5. Hudson’s (1954) description of seasonal cycles of Rubus idaeus L.
root cuttings

The “on” season for Rubus idaeus is from October to March. In contrast
there were months with little regeneration, which he called the intermediate
season: April, August, and September. An “off” season was one in which no

regeneration occurred from May through July. Further experiments by Hudson

13

 

(1955a) with horseradish (Cochlean‘a armoracia L.) and dandelion (Taraxacum
ofﬁcinale Weber ex Wiggers) demonstrated that not all plants follow a seasonal
cycle, but have strong root-bud regeneration year round. However, some growth
factors such as average number of shoots per cutting were affected by seasonal
changes. The “on” seasons described by Hudson occurred during and after
winter when cold was given to the root cuttings.

Evidence suggests that carbohydrate composition of roots has marked
effects on growth and development during the next growing season.
Carbohydrate reserves are important for the survival of the plant during the
winter and resumption of growth in the spring. These reserves are associated
with overwintering strategy in herbaceous perennials (Liu et al., 1993).

When rhubarb roots were forced before sufﬁcient cold treatment, plants
lacked vigor or did not grow (Fry, 1957). Low temperature treatment has been
described as a “starter” that needs to accumulate. Fry (1957) described rhubarb
plants that die back after cold as not having enough accumulated low
temperatures. In 1975, Rutherford and All described carbohydrate changes
during cold storage of rhubarb roots and found that rhubarb roots depend upon a
cumulative effect of cold. Carbohydrates in the crowns and roots of rhubarb
break down considerably (Rutherford et al., 1972). In general, cold storage
caused a breakdown of the insoluble polysaccharides, accompanied by a
corresponding increase in soluble sugars (Rutherford and Weston, 1968).
Similar studies in Panax ginseng revealed that the glucose content in roots is

higher in winter than spring, whereas starch content is lower (Liu et al., 1993).

14

This carbohydrate correlation with cold could explain Hudson’s “on” cycle that
coincided, in later experiments, with months of cold temperature and hydrolysis
of polysaccharides to sugars of lower molecular weight.

Similar ﬁndings have been published on kiwifruit root cuttings that
exhibited increased regeneration potential during winter and spring. Cuttings
taken at other times of the year exhibit poor regeneration (Lawes and Sim, 1980).
Robinson and Schwabe (1977a) observed similar seasonal ﬂuctuation in apples.

Generally, sugars move to the roots and are converted to polysaccharides
in autumn. Polysaccharides in the roots are then hydrolyzed during the winter,
leaving soluble sugars that translocate to root sprouts and accelerate spring
growth (Robinson and Schwabe, 1977b). Rutherford and Weston (1968) studied
polysaccharide changes in roots and tubers during cold. Their studies on tubers
of Jerusalem artichoke (Helianthus tuberosus L.) and roots of chicory (Cichorium
intybus L.) and dandelion showed that cold storage causes breakdown of
insoluble polysaccharides, accompanied by increasing soluble sugars of lower
molecular weight. Measurements of sprout height, performed by Robinson and
Schwabe (19773) on apple cultivars after one month of growth from root cuttings
showed that cold-stored roots grew faster than those without cold treatment.
However, after three months of growth, uncooled roots produced taller sprouts
than cooled ones. Rooting of detached sprouts was faster on roots placed in
cold storage for three months. Cold storage caused earlier rooting, suggesting

polysaccharide hydrolysis, since cold allows better availability of soluble sugars.

15

Carbohydrates and hormones like indoleacetic acid (IAA) play important
roles in shoot regeneration from roots of apple cultivars (Robinson and Schwabe,
1977b). Lawes and Sim (1980) suggested that a suitable balance between
carbohydrates, cytokinin, and auxins affects regeneration. Heuser (1977)
suggested that exogenously applied auxin and cytokinin could favorably
stimulate root-cutting production.

Hormonal control

Seasonal changes have an effect on lAA concentrations. High
concentrations cause root budding and inhibition of lateral stem shoots.
Hormones play an important role in root-bud proliferation. Root budding in apple
occurs only after roots are detached from the tree. Root cuttings exhibit polarity,
where shoots develop at the proximal end, the end nearest the stem; roots, at the
distal end, the end farthest from the parent plant, sometimes regeneration
occurred regardless of orientation (Ede et al., 1997). Furthermore, a polar
distribution of buds suggests a gradient of inhibition and promotion. Experiments
on kiwifmit showed better sprout regeneration on roots placed horizontally than
vertically, suggesting acropetal movement of auxins (Lawes and Sim, 1980).
Root-bud production appears to be hormonally controlled because some plants
produce root buds only after roots are detached from the mother plant. Robinson
and Schwabe (1977b) studied endogenous auxins and exogenous application of
auxins. Endogenous levels peaked during November, coinciding with the highest
regeneration potential and survival of root cuttings. Exogenous application of

IAA caused root-budding inhibition in apple cultivars, while 75 mg of benzylamino

16

purine (BAP; cytokinin) per liter increased proliferation to three to four times that
of control plants (Robinson and Schwabe, 1977b). Similar studies on dwarf
apple rootstocks (Kanazawa et al., 1978) and kiwifruit by Lawes and Sim (1980)
showed that sucrose and benzylaminopurine applied to the root cuttings caused
increased regeneration. lndolebutyric acid (IBA) application to the proximal end
Of the cutting caused inhibition of regeneration. Gibberellic acid and
Benzylaminopurine application increased the number of shoots per cutting.

Polar distribution of buds in root cuttings suggests a hormone gradient of
inhibition and promotion. Farmer (1962) working with, Populus tremuloides
Michx., visualized aspen roots as a “physiological” extension of stems and
observed that root buds on these roots develop when inhibiting inﬂuences were
removed. Farmer’s “apical dominance theory”, described as an extension of
apical dominance or correlative inhibition mechanism, was supported in his
publication by the stimulation of suckering by root pruning. Furthermore,
sprouting did not occur severed roots that were still attached to the stems. Maini
(1968) reported similar results of inhibition of regeneration while roots were still
attached to the mother plant. Correlative inhibition is a hormonal signal
mechanism whose mode of action is unknown and which controls lateral bud and
root-bud growth. Horvath’s (1998) studies with EuphOrbia esula L. showed
correlative inhibition of root buds required the presence of leaves and meristems.
The lack of root-bud emergence in E. esula was associated with an extension of
apical dominance caused by meristematically produced auxin and possibly an

unknown carbohydrate. Plants with a correlative inhibition mechanism tend to

17

produce reparative root buds when the roots are stressed or cut from the mother
plant, because the inhibition mechanism is removed. In some varieties of apples,
warmth causes a lower auxin concentration in cut roots, which promotes root
buds, because correlative inhibition does not function.

Correlative inhibition is also exhibited on root sprouts; they produce
hormones that can inhibit further bud initiation. Lindsay (1877) observed that,
regardless of the root-cutting length, the ﬁrst bud that usually regenerated in a
root cutting was the one that grew, inhibiting the remaining buds. When sprouts
are detached from roots, root-bud proliferation increases, probably because of
active regrowth from cut surfaces and removal of shoot inhibition (Robinson and
Schwabe, 1977a). Regenerated sprouts in a root cutting can more actively
inhibit other root buds from proliferating than gradual depletion of reserves
(Robinson and Schwabe, 1977b).

As expected in auxin regulated mechanisms, light appears to play an
important role. Ghani and Cahalan (1991) showed that most of the root sprouts
produced came from root parts exposed to light, which suggests a light-induced
auxin translocation to the buried distal end of the root-cutting.

Conclusion

Asexual propagation by root cuttings is a labor-intensive procedure. The
ﬂoriculture-perennial industry requires new methods Of root-cutting propagation
to achieve fast, reliable, and homogeneous material. The industry must be able
to quantify root cuttings produced per plant. Many of the plant requirements

described by Hudson in the 19503, plant capacity and performance are unknown

18

in many perennial species. This lack of knowledge can result in unsuccessful
root-cutting propagation, making it unpopular among growers. New asexual
propagation methods with the proper plant environmental requirements can

increase market production of root cutting—propagated plants.

19

References
Anonymous. 1882. Propagation of plants by root cutting. Garden 12:389.

Armitage, AM. 1997. Herbaceous perennial plants: A treatise on their
identiﬁcation, culture, and garden attributes. Varsity Press, Athens, Ga.

Bailey, L.H. 1978. Hortus Third. Macmillan, N.Y.

Bath, T. and Jones, J. 1994. The gardeners guide to growing hardy geraniums.
Timber Press, Portland, Or.

Bosela, M.J. Root sprouts in anemone
[Online] Available email:
chongjoa@msu.edu from
bosela@prairie.nodak.edu March 1, 1999

Bosela, M.J. and Ewers, PW. 1997. The mode of origin of root buds and root

sprouts in the clonal tree Sassafras albidum (Lauraceae). Amer. J. Bot.
84(11):1466-1481.

Chong, J.A., Heins, R., Clough, E., Cameron, A., and Carlson, W. 2000.
Forcing perennials—Species: Anemone hupehensis. Greenhouse Grower
Feb.: 66-74.

Dir, MA. and Heuser, CW. 1987. The reference manual of woody plant
propagation from seed to tissue culture. Varsity Press, Inc. Athens, Ga.

Dore, J. 1955. Studies in the regeneration of horseradish. Ann. Bot. 19:127-137.

Ede, F.J., Auger, M. and Green, T.G.A. 1997. Optimizing root cutting success in
Paulownia spp. J. Hort. Sci. 72 (2):179-185.

Farmer, RE. 1962. Aspen root sucker formation and apical dominance. Forest
Science. 82403-410.

Fisher, KB. 1961. Perennial plants from root cuttings. Plant Propagators’
Society 11:39-50.

Flemer, III, W. 1983. Propagating shade trees by cuttings and grafts. Combined
proceedings: International Plant Propagators‘ Society 32:569-579.

Forrester, J.W. 1968. Principles of Systems. 2nd prelim. ed. Wright-Allen Press,
Cambridge, Mass.

Fossel, PM 1998. Divide and multiply. Country J. 25(3):62-65.

20

Fry, W.G. 1957. Experiments with rhubarb for forcing. Expl. Hort. 1:8-11.

Ghani, A.K.M.O. and Cahalan, CM. 1991. Propagation of Prunus avium from
root cuttings. Forestry 64(4):403-409.

Graf, AB. 1992. Tropica, Color cyclopedia of exotic plants and trees. 4th ed.
Horowitz/Rae, Fairﬁeld, New Jersey.

Graham, ST 1990. Perennial garden plants, or, The modern ﬂorilegium: A
concise account of herbaceous plants, including bulbs, for general garden
use. Timber Press, Portland, Or.

Gregory, LE. 1965. Physiology of tuberization in plants (Tubers and tuberous
roots). Handbuch der Planzenphysiologie 15(1):1328-1354.

Heuser, CW. 1977. Factors controlling regeneration from root cuttings. Comb.
Proc. Annu. Meet. lnt. Plant Propag. Soc. 27:398-402.

Horvath, DP. 1998. The role of speciﬁc plant organs and polar auxin transport in
correlative inhibition of leafy spurge (Euphorbia esula) root buds. Can. J.
Bot. 76(7):1227-1231.

Hudson, JP. 1954. Propagation of plants by root cuttings: l. Regeneration of
raspberry root cuttings. J. Hort. Sci. 29:27-43.

Hudson, J.P. 1955a. Propagation of plants by root cuttings: II. Seasonal
ﬂuctuation of capacity to regenerate from roots. J. Hort. Sci. 30:242-51.

Hudson, J.P. 1955b. The regeneration of plants from roots. Rep. XIVth lntl. Hort.
Congr. 1165-1172.

Hudson, JP. 1956. Increasing plants from roots. Gardeners Chronicle
Gardening Illustrated. May 12, 1956:528-529.

Jelitto, L. and Schacht, W. 1995. Hardy herbaceous perennials. Timber Press,
Portland, Or.

Kanazawa, N., Miki, N., Okuno, I., Yukinaga, H., and Tomana, T. 1978. Studies
on the root cutting of East Malling VII dwarf apple rootstock. J. Japan.
Soc. Hort. Sci. 47(3):327-336.

Lawes, GS. and Sim, BL. 1980. Klwifruit propagation from root cuttings
Actinidia chinensis, New Zealand. New Zealand J. Expt. Agr. 8(3,4):273—
275.

Lindsay, R. 1877. Propagation of plants by root cuttings. The Garden 12:389.

21

Liu, M., Li, R., and Liu, M. 1993. Adaptive responses of roots and root systems to
seasonal changes. Environ. Expt. Bot. 33:175-188.

Macdonald, B. 1986. Practical woody plant propagation for nursery growers.
Timber Press, Portland, Or.

Madison, C. Trees born of ﬁre and ice.
[Online] Available
http://www.nwf.org/nwf/natlwildlaspen.html. February 18, 1999.

Maini, J.S. 1968. The relationship between the origin of adventitious buds and
the orientation of Populus tremuloides root cuttings. Bul. Ecol. Soc. Amer.
49:81-82.

McMillan, BR 1980. The propagation of plants from root cuttings. The
Plantsman 2:54-62.

Miller, DR. and Goodin, JR. 1976. Cellular growth rates ofjuvenile and adult
Hedera helix L. Plant Sci. Letters 7:397-401.

O’Rourke FL. 1951. The effect ofjuvenility on plant propagation. Michigan State
College, Proc. Pla. Prop. Soc. 1:33-37.

Perry, LP. 1998. Herbaceous perennial production: A guide from propagation to
marketing. Northeast Regional Agr. Engineering Service, Cooperative
Extension.

Peterson, R.L. 1975. The initiation and development of root buds, pp. 125-161.

In: The development and function of roots, 3rd Cabot symposium. Edited
by J. G. Torrey and D. T. Clarkson. London; New York: Academic Press.

Robinson, J.C. and Schwabe, W.W. 1977a. Studies on the regeneration of apple
cultivars from root cuttings. I. Propagation aspects. J. Hort. Sci. 52:205-
220.

Robinson, J.C. and Schwabe, W.W. 1977b. Studies on the regeneration of apple
cultivars from root cuttings. ll. Carbohydrate and auxin relations. J. Hort.
Sci. 52:221-233.

Rutherford, PP. and Ali, NA. 1975. Carbohydrate changes during cold storage
of different cultivars of virus free and virus infected rhubarb. J. Hort. Sci.
50(3):249-255.

Rutherford, PP. and Weston, E.W. 1968. Carbohydrate changes during cold
storage of some inulin-containing roots and tubers. Phytochemistry

22

7:175-180.

Rutherford, P.P., Sewell, AP, and Case, MW. 1972. Carbohydrate changes
during the cold storage Of rhubarb roots, cultivar Victoria. Expt. Hort.
24:37-42.

Sanchez, J.H. 1999. Propagating from root cuttings. Horticulture 96(6):26-27.

Stoutemyer, VT 1968. Root cuttings. Plant Propagator 424-6.

Whyte, RC. 1938. Phasic Development of Plants. Biol. Rev. 14:51-87.

Wilson, B.F. 1975. Distribution of secondary thickening in tree root systems, pp.
197-219. In: The development and function of roots, 3rd Cabot

symposium. Edited by J. G. Torrey and D. T. Clarkson. London; New
York: Academic Press.

Wobst. 1868. Vennehrung der Pﬂanzen durch Wurzelstecklinge. Gartenﬂora
1 72292-296.

 

23

SECTION II

EFFECTS OF PHOTOPERIOD AND COLD TREATMENT ON DRY
WEIGHT PARTITIONING OF ANEMONE HUPEHENSIS AND ANEMONE
XHYBRIDA, AND A ROOT-PLUG METHOD FOR PROPAGATION OF
ROOT CUTTINGS.

24

Effects of Photoperiod and Cold Treatment on Dry Weight Partitioning of
Anemone hupehensis and Anemone xhybrida, and a Root-Plug Method for

Propagation of Root Cuttings.

Joaquin A. Chong‘, Royal Heinsz, Arthur C. Cameronz, and William Carlson2

Department of Horticulture, Michigan State University, East Lansing, MI 48824-1325

Received for publication . Accepted for publication . We
gratefully acknowledge the ﬁnancial support of the Agricultural Experiment
Station of Michigan State University and funding by producers and greenhouse
growers supportive of Michigan State University ﬂoricultural research.

1 Graduate Student.

2 Professor.

25

Effects of Photoperiod and Cold Treatment on Dry Weight Partitioning of
Anemone hupehensis and Anemone xhybrida, and a Root-Plug Method for
Propagation of Root Cuttings.

Additional index words. Critical photoperiod, root bud, asexual propagation.

Abstract

The effects of photoperiod and cold treatment on dry-weight partitioning
of Anemone hupehensis (Thunb.) Bowels & Steam and A. xhybrida (Paxt.)
were determined and a root-plug method for asexual propagation of root
cuttings was developed and tested. Either before or following a 15-week
treatment at 5°C plants were grown under 10, 12, 13, 14, 16, or 24 hours of
continuous light or 9 hours with a 4-hour night brake lighting period. Shoot
and root dry weights were obtained after 16.5 or 15 weeks of growth for A.
hupehensis and A. xhybrida respectively; following either 0 or 15 weeks of cold
for A. xhybrida. Shoot-dry-weight partitioning was greater on reproductive
than vegetative plants. Root-dry-weight partitioning decreased with increasing
photoperiod and following cold. Cold increased root-buds and reduced root-
bud appearance time. A maximum of 60% and a minimum of 32% of total dry
weight was allocated to roots under 10-hr photoperiods without cold or 24-
hour—photoperiods with cold, respectively.

The root-plug experiment was conducted to test a new method for more
efﬁcient root-cutting propagation. A root-plug (RP) technique was developed
that consisted of growing mother plants in a 1.5 litter container with internally
divided compartments, speciﬁcally a 128-cell-plug tray above a 288-cell-plug
tray or three 288-ceIl-plug trays horizontally or vertically oriented. Neither
tested plant age (7.5- or 28-week) nor cold treatment (0- or 15-week) affected
root plug regeneration. Top trays produced the most RPs, up to an average of
17.3 per 25 cells. Root-plug production in plug trays decreased with
increasing distance from the crown. Vertically oriented trays suggested that
roots had to elongate to thicken as middle trays produced more RPs than side
trays.

26

Introduction

Asexual propagation allows clonal propagation and perpetuation of
germplasm. Sterile plants only can be propagated asexually, sometimes by
root cuttings. Macdonald (1986) described root cutting as a technique in which
roots capable of developing root buds (shoots, suckers) are cut into individual
pieces capable of regenerating plants. Adventitious buds develop from roots
that have been cut into individual pieces and planted upright in a growing
medium. Root-cutting propagation depends on the ability of certain roots to
produce root buds that subsequently form shoots.

Root buds are deﬁned as subterranean adventitious shoot buds located
on lateral roots (Horvath, 1998). Hudson (1955) described bud-producing
roots as a piece (2 to 3 cm) of secondary thickened root capable of growing
buds during a certain season. Roots must be mature or have reached the
“ripeness to regenerate” developmental phase (Dore, 1955), before they are
capable of root-bud production. A mature root has a primary cortex and has
developed some secondary tissue.

More than a century ago, Wobst (1868) suggested that better
regeneration from root cuttings occurred when cuttings were taken in early
spring before the mother plant developed. An understanding of the
mechanisms behind this empirical knowledge did not start until the mid-19508
when Hudson published several scientiﬁc papers about the effects of seasonal
ﬂuctuations on root regeneration, growth, and development. Hudson (1954)

classiﬁed months of good regeneration as “on” season, months with little

27

regeneration as intermediate season, and months with no regeneration as “off”
season. He also found that not all species followed a seasonal cycle (Hudson,
1955)

Later studies suggested that carbohydrate composition of roots had
marked effects on growth and development of root cuttings (Robinson and
Schwabe, 1977b). Controlling the partitioning of carbohydrates becomes
important for proper root-cutting development. Larger carbohydrate reserves
can increase survival of plants during winter and promote growth resumption in
the spring. Reserves are associated with the overwintering strategy in
herbaceous perennials (Liu et al., 1993). Reserves partition between shoots
and roots and changes in partitioning are probably stimulated by photoperiod
and the plant developmental phase. In potato, for example, it is generally
agreed that short days (SD) promote tuberization and long days (LD) delay it
(Gregory, 1965). A common parameter for evaluating partitioning between
shoots and roots is the shoot : root ratio (Wolfgang, 1979) or, inversely, the
root : shoot ratio.

Generally, sugars move to the roots and are converted to
polysaccharides in autumn. The polysaccharides are then hydrolyzed during
the winter, leaving soluble sugars that translocate to root sprouts and
accelerate spring growth (Robinson and Schwabe, 1977b). Rutherford and
Weston (1968) studied polysaccharide changes in roots and tubers exposed to
cold. Their studies on tubers of Jerusalem artichoke (Helianthus tuberosus L.)

and roots of chicory (Cichon’um intybus L.) and dandelion showed that cold

28

storage causes breakdown of insoluble polysaccharides, accompanied by an
increase in soluble sugars of lower molecular weight.

Polar distribution of buds in root cuttings suggests a hormone gradient
of inhibition and promotion. Farmer (1962) working with Populus tremuloides
Michx., visualized aspen roots as a “physiological” extension of stems and
observed that root buds on these roots develop when inhibiting inﬂuences
were removed. Farmer's “apical dominance theory” is described as an
extension of apical dominance or a correlative inhibition mechanism was
supported in his publication by the stimulation of suckering by root pruning.
Furthermore, sprouting did not occurred on severed roots that were still
attached to the stems. Maini (1968) reported similar results of inhibition of
regeneration while roots were still attached to the mother plant. Correlative
inhibition is a hormonal signal mechanism that controls lateral bud and root-
bud growth and whose mode of action is unknown.

Correlative inhibition is also exhibited on root sprouts: they produce
hormones that can inhibit further bud initiation. As early as 1877, Lindsay
Observed that, regardless of the root-cutting length, the ﬁrst bud that
regenerated in a root cutting was the one that grew ﬁrst, and it inhibited the
remaining buds. Root-bud proliferation increases when ‘sprouts are detached
from roots, probably because of active regrowth from out surfaces and removal
of shoot inhibition (Robinson and Schwabe, 1977a). Regenerated sprouts in a
root cutting can more actively inhibit other root buds from proliferating than

gradual depletion of reserves (Robinson and Schwabe, 1977b).

29

Root-cutting propagation is important when the plant has difﬁcult-to-root
stems, does not bear seed, or does not produce seed that is true to type
(Fisher, 1961; Macdonald, 1986). Root cuttings can eliminate the need for
rootstocks, budding, and grafting (Robinson and Schwabe, 1977a). Root
cuttings might have a longer life than grafted clones because grafting might
cause scion understock incompatibilities (Flemer, 1983). There are several
disadvantages to root-cutting propagation. Root cuttings have been described
as one of the least frequently used and least desirable methods of vegetative
propagation (Fisher, 1961; Flemer, 1983; Heuser, 1977) for several reasons.
Root-cutting propagation is tedious and messy. Root cuttings are more
difﬁcult to gather than aerial cuttings (Flemer, 1983). The method is labor
intensive, the plants must be dug or the soil excavated to expose the roots,
and the procedure must be carried out during the fall or winter (Heuser, 1977).
There might be yearly differences in root material. In some years it might be
difﬁcult to obtain large quantities of root-cutting material (Ede et al., 1997;
Flemer, 1983). Chimeric material might not come true to type if the mutated
section is not propagated or if the mutation is in the shoot section. Little
research has been aimed at the quantiﬁcation of root cuttings per plant
(Macdonald, 1986). Without knowledge of seasonal effects on the roots, it is
impossible to morphologically distinguish roots capable of producing root buds
(Hudson, 1954). Finally, there is a general uncertainty about and unfamiliarity

with this procedure.

30

Anemones can be propagated by seed, root cuttings or division (Perry,
1998). Seed propagated anemones are variable and not true to type.
Arrnitage (1997) described anemone root-cutting propagation as more
effective than shoot division propagation. Root cuttings are used as the main
source of commercially propagated material even though root-cutting
propagation varies yearly and protocols are unavailable.

The goal of our research was to understand how photoperiod and cold
treatment affect anemone dry-weight (DW) partitioning and control these
factors to increase root mass and root-bud regeneration. Stimulating root
mass or dry weight partitioning to roots can facilitate root-bud regeneration as
larger root reserves can improve regeneration (Robinson and Schwabe,
1977a). The second goal of our research was to develop a procedure to

facilitate, accelerate and homogenize asexual propagation by root cuttings.

Materials and methods

Plant material

Dry Weight Partitioning Experiment. Asexually propagated Anemone
hupehensis and A. xhybrida ‘Whirlwind’ plants arrived in 36-cell plug trays from
a commercial greenhouse (Greenleaf Enterprises, Inc., Leola, Penn.) on
September 17, 1998, and on November 11, 1998, respectively. Plants varied
in size and number between plug cells and were therefore singulated.

Root-plug Experiment. Anemone xhybrida ‘Whirlwind’ and Anemone

hupehensis were propagated by root cuttings from one-year-old plants

31

received in one-gallon containers on June 18, 1998. When propagated, all
plants from root cuttings were planted in 32-cell plug trays (130 mL per cell)
from Summit Plastic Co. (Tallmadge, Ohio) in the same medium used in the
DW partitioning experiment.

Plant culture

Plants were top-watered and fertilized at every irrigation throughout all
experiments. The nutrient solution was well water (EC of 0.70 mS-cm"1 and
105, 35, and 23 mg Ca, Mg, and SH, respectively; pH 6.0) acidiﬁed with
H2804 to a titratable alkalinity of 130 mg CaCO3-L'1and water soluble
fertilizer providing 125-12-125-13 N-P-K-Ca (mg-L4; 30% ammoniacal N) plus
1.0, 0.5, 0.5, 0.5, 0.1, and 0.1 Fe, Mn, Zn, Cu, B, and MO, respectively (mg-L"
1; MSU Special, Greencare Fertilizers, Chicago, 1".)

Dry weight partitioning experiment

In the DW partitioning experiment, plants were randomly divided into
two groups, a 0- and 15-week cold treatment. Uncooled plants were
transplanted to 13-cm square plastic containers (1.1 L) ﬁlled with a commercial
soiless medium composed of pine bark, vermiculite, Canadian sphagnum
peat, and coarse penite with a wetting agent, lime, and starter fertilizer charge
(High Porosity Mix, Strong-Lite Products, Pine Bluff, Ark.) and moved to
photoperiod treatments.

Photoperiods were provided by pulling opaque black cloth from 1700

until 0800 HR every day over all benches; incandescent lamps (z2 umol-m'Z-S‘

1) were turned on to complete each photoperiod. Ten plants were placed per

32

bench and incandescent lights treatments set to complete 10-, 12-, 13-, 14-,
16- or 24-h photoperiods as day extension, and one was set as a 4-h night
interruption (NI) from 2200 to 0200 HR.

Cold consisted in 15 weeks of 5 °C in a cooler. After cold treatment,
plants were transplanted to the 13-cm square plastic containers (1.1 L) and
placed under photoperiods as described for plants without cold.

Root-plug experiment

Two cultivars were tested in the root-plug (RP) experiment, Anemone
xhybrida and A. hupehensis. The A. xhybrida experiment was a factorial with
three factors: plant age, tray treatment and cold. For A. xhybrida, two plant
ages were tested; plants propagated on September 21, 1998, or February 12,
1999. At the start of the experiment on April 6, 1999, all plants propagated on
September 21 had trilobate compound leaves, and plants propagated on
February 12 had monolobate simple leaves.

Plants were planted in 1.5-L square black containers 13 cm wide and
13 cm tall (Super Square, National Polymers, lnc., Lakeville, Minn.) with plug
trays cut to ﬁt inside each pot (Fig. 1). Two trays sizes were tested, a 288-cell
plug tray consisting in 2-cm-wide and 2.6-cm-tall cells with 6 mL per cell, and a
128-cell plug tray consisting in 2.7-cm-wide and 5-cm-tall cells with 25 mL per
cell (Blackmore CO., Belleville, Mich.)

The tray-treatment factor for A. xhybrida plants had two levels: the ﬁrst
had one cut-tO-ﬁt 128-cell plug tray set above a cut-to-ﬁt 288-cell plug tray

inside the pot (128l288 treatment; Fig. 1A). The top 128-cell tray had nine

33

complete cells and seven half cells, and the bottom 288-cell plug tray was cut
to ﬁt in the bottom of the container, leaving 20 complete cells and ﬁve half
cells. The second treatment had three 288-cell plug trays stacked horizontally
inside the container (288/288/288 treatment; Fig. 1B). The top two trays had
25 complete cells; the bottom tray, 20 complete and ﬁve half cells. The
bottoms of all 128-cell plugs were cut off in the 128/288 treatment, and
drainage holes of the top two 288-cell plugs trays in the 288/288/288 treatment
were enlarged with a 1-cm ﬁle to allow root growth into the next trays.
Photoperiod for the RP experiment remained at 16 h throughout the

experiment and was provided by day extension lighting at a minimum of 50
umol-m'zs1 photosynthetic photon ﬂux (PPF) and an average of 65 umoI-m'

2-3'1 at plant height with high-pressure sodium (HPS) lamps from 1700 to
2400 HR.

The cold factor had two levels, 0 and 6 weeks. After 15 weeks of plant
growth from the transplant day (April 6), half of the A. xhybrida and A.
hupehensis plants were transferred to a 5 °C controlled-environment chamber
for 6 weeks. The other half was cut to determine shoot DWs; for the cooled
plants, they were determined after cold. The roots and plug trays were
removed from the 13-cm container, and plug trays were cut with a sharp knife
to separate them from the plant soil mass and trays. Plug trays were placed
under the 16-h photoperiod for root-bud formation. Root plugs regenerated

after 5 weeks of growth were counted on each tray.

34

The A. hupehensis experiment was a factorial with two factors: tray
treatment and cold. The A. hupehensis experiment started on June 9, 1999,
with plants propagated by root cutting on March 3, 1999. The tray treatment
factor had three levels, the ﬁrst two as previously described for the A. xhybrida
experiment. The third level, 288-288/288 treatment (Fig. 1C), consisted in
using three 288-cell cut-to-ﬁt plugs trays placed vertically inside a container.
The plants were transplanted between a 288-cell tray and two, 288-cell
vertically placed trays, bottoms to the sides. The drainage holes of the 288-
cell center plug tray were enlarged with a 1-cm ﬁle to allow roots to grow to the
adjacent tray.

Tray treatments occupied z0.9 L of the volume of the container, leaving

0.6 L where the plant was transplanted. Trays and containers were ﬁlled with
a commercial soilless medium composed of peat (70%), coarse perlite (21%),
vermiculite (9%), a wetting agent, lime (calcitic and dolomitic), and a starter
fertilizer charge (Suremix Perlite, Sure Michigan Growers Products, Inc.,
Galesburg, Mich.)

Experimental settings

All experiments were conduced in the Plant and Soil Sciences
Research Greenhouses at Michigan State University, East Lansing, Michigan.
Greenhouse temperatures were set the same for DW and RP experiments: 20
°C. The average daily temperature (ADT) and daily light integral were
monitored with a CR-10 datalogger (Campbell Scientiﬁc, Logan, Utah) by

using 36-gauge type-E thermocouples and quantum sensors (Ll-COR),

35

respectively. The datalogger collected temperature and light measurements
every 10 s and recorded the hourly average. Under each black cloth in the
DW experiment, heat was provided when needed to maintain a night
temperature of 20 °C by using a 1500-W electric heater (Model T771, Rival
Manufacturing Co., Sedalia, Mo.)

Supplemental lighting for both the DW and RP experiments was

switched on from 0800 to 1700 HR if the ambient greenhouse PPF was below
200 leOI'm'2°S'1 and switched off if it exceeded 400 umol-m'Z-S'I. Four-
hundred-watt HPS lamps provided a minimum of 50 pmol-m'Z-S'1 PPF and an

average of 65 umoI-m'Z-S'1 at plant height.
Lighting in the cooler was the same for both experiments and was from
cool-white ﬂuorescent (F96T12/CWNHO, Philips, Somerset, NJ.) lamps from

0800 to 1700 HR. The PPF from the cool-white ﬂuorescent lamps was z100

umoI-m'Z-S'1 at plant height. Plants in the chamber were watered as needed
(approximately two times per week) with well water acidiﬁed with sulfuric acid

(H2804) to a pH of approximately 6.0.

Data collection

Dry weight partitioning. Dates the root bud was ﬁrst visible were
recorded approximately three times a week for A. xhybrida. Root buds were
considered ﬁrst visible when they could be seen in soil mass after it was lifted
from the container. Shoot and root DWs were obtained after 15 weeks Of
forcing for ‘Whirlwind’ and 16.5 weeks for A. hupehensis. Shoots and roots

were dried for 3 days in a NAPCO model 630 oven (National Appliance Co.

36

Portland, Ore.) at a setting of 65 °C. For A. x hybrida, all the soil was
separated from the roots before plants were dried, and root buds longer than
0.75 cm were counted. The DW experiments were analyzed as a factorial
design with two factors, cold and photoperiod treatment: cold had two levels
and photoperiod has seven levels. Ten plants per treatment were used and
data were analyzed with SAS’s (SAS Institute, Cary, NC.) analysis of variance
(ANOVA) and a general linear model (GLM) procedure.

Root-Plug Experiment. Shoot DWs were obtained from mother plants
after 15 weeks of forcing for plants not treated with cold and after 6 weeks for
cold-treated mother plants. Number of regenerated RPs per tray level was
counted 5 weeks after cutting.

The A. xhybrida and A. hupehensis experiments were analyzed as a
three- and two-way factorial for DWs, respectively. Dry weights, total RPs,
and weeks of cold were analyzed with SAS’s (SAS Institute, Cary, NC)
ANOVA and a GLM procedure. Tray levels were analyzed separately with

SAS’s mixed procedure, repeated variables with a compound symmetry type.

Results

Dry weight partitioning experiment

Anemone hupehensis. Dry weight partitioning was affected by
photoperiod. Root DW decreased when photoperiod exceeded 14 h, the same
photoperiod that induced ﬂower formation (Fig. 2A). Shoot DW, however,

increased with increasing photoperiod, with the greatest increase occurring from

37

the 10- to 14-h photoperiod treatment. Plant variability as shown by 95%
conﬁdence intervals was greater for shoots than for roots. The maximum visible
ﬂower bud percentage was 60 under the 24-h photoperiod. Dry weight root :
shoot ratio linearly decreased statistically with increasing photoperiod from 10 to
24 h, although most change occurred from 10- to 16-h photoperiod (Fig. 28).

Anemone xhybrida. As for A. hupehensis (Fig. 2A), not all uncooled A.
xhybrida plants ﬂowered, even under continuous light (Fig. 3A). All plants cold-
treated for 15 weeks initiated ﬂowers under 314-h photoperiods or NI. Dry weight
in the uncooled plants increased lineaiiy with increasing photoperiod from 10 to
24 h. Dry weight for plants cold-treated for 15 weeks increased from the 10- to
13—h photoperiod, then decreased (Fig. 3B). The 14-h photoperiod data point
was excluded from the analysis, since plants were closer to the cooling pads and
were apparently affected by the lower greenhouse temperature. The root : shoot
ratio for plants with a cold treatment was lower than for plants without cold (Fig.
3C), however, cold treated plants had grater total DW (Fig. 3A and 3B), up to
13.8 9, except for 24-h photoperiod plants.

Without cold, DW partitioning to roots decreased as photoperiod increased
from 10 to 14 h (Fig. 3C) and then remained the same. Following cold, root DW
partitioning was least favored by photoperiods longer than 12 h. The root : shoot
ratio decreased linearly as photoperiod increased from 10 to 24 h.

Root bud production appears earlier under 13-h photoperiod cold treated
plants, however no horticulturally important trends were shown (Fig. 4A). For

cold treated plants, root-bud number was highest in the 10- to 12-h photoperiod,

38

then decreased as photoperiod increased (Fig. 48). Root bud numbers were
similar across all photoperiods for uncooled plants. Plant height increased
linearly for cold treated and untreated plants as photoperiod increased (Fig. 40).
Cold treated plants were taller than uncooled plants.

Root-plug experiment

Anemone xhybrida. Shoot DW for plants grown for 15 weeks or 15 weeks
following 6 weeks of cold averaged 30.4 g and was not signiﬁcantly different for
plant age or tray treatment (data not shown). Weeks of cold did not signiﬁcantly
affect total RPs produced for any tray level: therefore, data were pooled for
further analysis. Total RPs per 0.9 L container volume was calculated to assess
RPs produced per volume for both tray treatments (Fig. 5A and 5B). In the
128/288 treatment (Fig. 5A), average RP production was 10.6 and 12.1 for the
7.5- and 28-week-old mother plants, respectively. The 7.5-week-old plants in the
288/288/288 treatment (Fig. 5B) produced an average of 27.5 RPs, with the 28-
week-old plants producing an average of 11.4 RPs.

The most RPs produced in both plug tray treatments were in the top level
(Fig. 5C and 5D). In the 128/288 treatment, average RP production was a
statistically similar 10.3 and 11.8 for 7.5- and 28-week mother plants,
respectively. The top, middle, and bottom of the 7.5-week-old plants in the
288/288/288 treatment produced an average of 17.3, 7.7, and 0.5 RPs,
respectively (Fig. 5D). Few or no RPs were produced at the bottom levels. For
the 28-week mother plants, the average RP production was 9.4, 2.0, and 0, for

top, middle, and bottom trays, respectively (Fig. 5D).

39

Anemone hupehensis. Only one plant age was tested for A. hupehensis.
Total RP production in the 128l288 treatment averaged 9 (Fig. 6A); in the
288l288/288 treatment, 13.5 (Fig. 6B). As with the A. xhybrida experiment,
upper levels produced the most RPs, averaging 8.5 and 10.3 for top levels of the
1281288 and 288l288/288 treatments, respectively.

The 288-288/288 treatment produced a combined average of 9.4 RPs for
all levels (Fig. 6C). The left, middle, and right trays averaged 2.2, 5.2, and 2.0

RPs, respectively.

Discussion

Dry weight partitioning experiment

In 1955, Hudson stated that root cutting behavior depended on two
factors, plant capacity and plant performance. Plant capacity refers to required
factors required for regeneration success before root cuttings are taken from the
mother plants.» Plant performance refers to the environmental and hormonal
factors that affect root cuttings after the roots are separated from the mother
plant. The purpose of the DW partitioning experiment was to understand how
photoperiods and cold treatment affect A. hupehensis and A. xhybrida root-to-
shoot DW partitioning, a plant capacity issue. Dry weight partitioning between
roots and shoots can help us identify the optimal environmental conditions for
mother-plant growth. Larger root DWs are synonymous with larger storage
reserves, which presumably mean more photosynthates that increase root-bud

regeneration (Robinson and Schwabe, 1977a).

40

The data showed that photoperiod and cold inﬂuenced DW partitioning
between shoots and roots. The data suggested that as the plant enters its
reproductive development, plant shoots and ﬂowers become strong sinks and
reduce partitioning to the roots. In general, photoperiods above 14 h caused
bolting, ﬂowering, and the shift of DW partitioning to the ﬂower producing organs
(Fig. 2A, 23; Fig. 3A, 3B, 30; Fig. 40). According to data from 15- and 16.5-
week-old plants, the best photoperiod for root weight partitioning is under short,
noninductive photoperiods. It is possible, however, that a longer growth duration
under LD could yield more root DW than under SD, because less photosynthates
are produced during short days (Gregory, 1965).

Anemone has been classiﬁed as a cold-beneﬁcial LD plant; i.e., after a
cold treatment, plant growth is more vigorous (E. Clough, unpublished
data). Cold generally decreased DW partitioning to roots and increased DW of
shoots (Fig. 3C).

Cold increased visible ﬂower-bud percentage (Fig. 3B) and increased the
number of root buds formed per plant under photoperiods shorted than 12 h (Fig.
48). Root bud formation on anemone does not appear to be directly controlled
by a correlative inhibition mechanism, as Maini (1968) described, but rather
conforms to Hudson’s (1955) description of bud-producing roots, any piece (2 to
3 cm) of secondary thickened root capable of growing and developing during a
certain season. In this experiment, regenerated root buds were produced from

roots z0.2 mm or larger in diameter.

41

Root-plug experiment

The purpose Of the RP experiment was to devise a new method of root-
cutting propagation to ease root handling, avoid polarity problems, and make
propagation more efﬁcient and quantiﬁable. As expected, more regeneration
occurred closer to the crown where the roots were thicker (Fig. 50, 5D, GD and
6E), results in agreement with those of other studies (Dir and Heuser, 1987;
Ghani and Cahalan, 1991; Robinson and Schwabe, 1977a; Wilson, 1975). The
plant crowns sat on the ﬁrst tray inside the pots, except for those in the 288-
288/288 tray treatment, and as roots grew into the top plug trays, their ability to
get into middle or bottom trays was reduced because of the funneled architecture
of the trays. This architecture forced some roots to grow alongside the plug
trays; these roots expanded freely to the bottom of the container, where
secondary thickening occurred, and some were capable of root-bud production.
The need for expansion for root thickening is suggested by the 288-288/288
treatment (Fig. 6F) in which the middle tray produced more RPs than the left tray,
presumably because the center tray‘s could expand to the right tray. To avoid
the root funneling problem, we are conducting another experiment in which
plants are grown over light egg crates that are positioned vertically and
horizontally (unpublished data) under a 13- and 16-h photoperiod.

Root plugs can be deﬁned as plantlets produced from roots that grow into
internal divided compartments inside a container; the compartments are later cut
into layers and root buds are regenerated from them. Root plugs might prevent

polarity problems; increase time efﬁciency; homogenize the crop, since root buds

42

are regenerated at the same time; reduce root handling; and help quantify the
plantlets produced per pot. However, there is a need for a manufactured
container with internal unfunneled divisions that would facilitate the setup of the
mother plant transplanting.

In conclusion, as any asexual propagation, anemone root-cutting
propagation requires disease free mother plants preferably grown over a season
to increase root thickness. For industry production plugs should be uniform and
homogeneous root cuttings should preferably be regenerated at the same time or
sorted according to size when separated form the root mass. Although, short
photoperiods can increase root dry weight partitioning, long photoperiods without
cold increased total root mass (Fig. 3A). Future investigation shall include a
combination of weeks of long then short photoperiods, which might increase root
mass and root partitioning, respectively. Other possibilities might include spray
applications of ethephon to chemically remove ﬂower buds and force a sink

partitioning to roots.

43

References

Armitage, AM. 1997. Herbaceous perennial plants: A treatise on their
identiﬁcation, culture, and garden attributes. Varsity Press, Athens, Ga.

Dir, MA. and Heuser, CW. 1987. The reference manual of woody plant
propagation from seed to tissue culture. Varsity Press, Inc. Athens, Ga.

Dore, J. 1955. Studies in the regeneration of horseradish. Ann. Bot. 19:127-137.

Ede, F.J., Auger, M. and Green, T.G.A. 1997. Optimizing root cutting success in
Paulownia spp. J. Hort. Sci. 72(2):179-185.

Farmer, RE. 1962. Aspen root sucker formation and apical dominance. Forest
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Fisher, KB. 1961. Perennial plants from root cuttings. Plant Propagators’
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Flemer, HI, W. 1983. Propagating shade trees by cuttings and grafts. Combined
proceedings: Int. Plant Propagators' Soc. 32:569-579.

Ghani, A.K.M.O. and Cahalan, CM. 1991. Propagation of Prunus avium from
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Gregory, L.E.1965. Physiology of tuberization in plants (Tubers and tuberous
roots). Handbuch der Planzenphysiologie 15(1):1328-1354.

Heuser, CW. 1977. Factors controlling regeneration from root cuttings.
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Hudson, JP. 1954. Propagation of plants by root cuttings: l. Regeneration of
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Hudson, JP. 1955. Propagation of plants by root cuttings: ll. Seasonal
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Timber Press, Portland, Or.

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Robinson, J.C. and Schwabe, W.W. 1977a. Studies on the regeneration of apple
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Wolfgang, B. 1979. Methods of studying Roots Systems. Springer-Veriag, Berlin;
New York.

45

 

A 128/288

 

128

V\7i”/\ /\

 

 

 

 

288l288/288

 

 

 

 

 

 

 

 

 

 

 

 

Figure 1. Drawing of root-plug tray treatments for Anemone hupehensis (A and
B) and A. xhybrida (A, B and C) root-plug experiment. Tray treatments 128l288,
288/288/288, and 288-288/288 are represented in ﬁgures A, B, and C,
respectively.

46

 

- 1OO

 

16- ______________ _T _

 

)

 

(

 

Dry weight (g)
S

 

 

 

 

I-8O

~60

-40

-20

 

 

 

1.5

1.0. III_ _

.____——__ _—

Root 2 shoot ratio

 

 

L** QNS g;
0.5 -

 

 

10 12 14 16 18 20
Photoperiod (h)

Figure 2. A: The effects of photoperiod on Anemone hupehensis root (unﬁlled
circles) and shoot (ﬁlled circles) dry weights and visible ﬂower-bud percentage

<--)

(%) pnq Jemou eICIISIA

(unﬁlled diamonds). B: Dry root to shoot ratios in response to photoperiod. Error

bars are 95% conﬁdence intervals and for clarity are offset to the right of data
points for shoot dry weights. L = linear; Q = quadratic trends. ”3'“ Nonsigniﬁcant

or signiﬁcant at P 5 0.01.

47

 

 

 

 

 

 

  
    

 

 

 

 

 

 

 

 

 

 

A Oweeks of cold
+ Leaves _ 100
15 —0— Roots %
3 ‘ 6‘
E - 80 5
:3
c: . o
.- " 60 |
3 1° : 5
2‘ - 40 V c'
D a
5 - - 20 §
——4—<>-—t—-<>—<>—<> : t e i : F r i : 4 :
B 15 weeks at 5 °C
_____ —O O "" 100 <
A 15 . _____ ‘2’:
C” _ 0'
:: 8° 5
'5) l ’r 3”
"7’ 10 l 4- ’ 6° : 2
3 l 1 T 9.
a f . 40 V 0'
5 - ——————————— / 1 20 ’03
—o-—<>—4——<) : L 5 g L
C
.2 2.0 -- - -—
’é
*-' 1.5 - ------
8
.5 1
II 1.0 — ———————————— - r
‘5 i
o
‘1 0.5 * -----------------
15 weeks at5°C L Q
0.0 .4 t : t .4 : : 2 : ; e

 

10 12 14 16 18 20 22 24M
Photoperiod (h)

Figure 3. The effects of photoperiod on the growth of Anemone xhybrida
‘Whirlwind’. A: Dry weight of uncooled plants and visible ﬂower bud percentage
measured at 15 weeks. B: Dry weight of plants cooled for 15 weeks and visible
ﬂower bud percentage. In A and B ﬁlled circles represent shoot dry weight;
unﬁlled circles root dry weight. C: Unﬁlled squares represent root 2 shoot ratios
of uncooled plants; ﬁlled squares, root 2 shoot ratios of plants cooled 15 weeks.
Error bars are 95% conﬁdence intervals and for clarity are offset to the right. L =
linear; Q = quadratic trends. NS'*'*** Nonsigniﬁcant at P 5 0.05 or 0.001

48

 

 

 

 

 

 

 

Days to visible root bud

 

(A)
O

 

N
O

 

 

_L
O

 

Number of root buds
longer than 0 75 cm

 

O)
O

 

.h
01

 

 

00
O

 

Plant height at
15 weeks (cm)

 

.A
(II

 

 

 

# L

 

10 12 14 16 18 20 22 24Nl
Photoperiod (h)

Figure 4. The effects of photoperiod on days to visible root bud (A), root bud
number (B), and canopy height (C) of Anemone xhybrida ‘Whirlwind’. Unﬁlled
symbols represent uncooled plants grown for 15 weeks. Filled symbols represent
plants after 15 weeks of 5 °C and 15 weeks’ growth. Error bars are 95%
conﬁdence intervals and for clarity are offset to the right of data points for 5 °C
treated plants. L = linear; Q = quadratic trends. NS'*'*** Nonsigniﬁcant or
signiﬁcant at P 5 0.05 or 0.001, respectively.

49

 

50 A 128/288 tray treatment B 288/288l288 tray treatment

0

Total root plugs produced
per treatment

 

 

 

 

_ - TOP 128 cell 0 O — TOP 288 cell
9 20 - 1:; BOT 288 cell MID 288 cell
2 :1 BOT 288 cell
83
O.
8 15 - o
o
3
'0
g
a, 10 —
O)
3
a
T5
0 -
m 5
O O
o _
7.5 Wk 28 Wk

 

Mother plant age

Figure 5. The regeneration of Anemone xhybida cut after 15 weeks of growth
and measured after 5 weeks of root-bud regeneration. Total root plugs produced
from 128/288 (A) and 288/288l288 (B) tray treatments. Root plugs produced per
level from 128/288 (C) and 288/288l288 (D) tray treatments. Error bars are 95%
conﬁdence intervals. Unﬁlled circles represent raw data and for clarity are offset
to the right when the same value is repeated.

50

Total root plugs produced
per treatment

Root plugs produced per level

Figure 6. The regeneration of Anemone hupehensis cut after 16.5 weeks of
growth and measured at 5 weeks of root-bud regeneration.
produced from 128/288 (A), 288/288l288 (B), and 288-288l288 (C) tray
treatments. Root-plugs produced per level from 128/288 (D), 288/288l288 (E),
and 288-288l288 (F) tray treatments. Error bars are 95% conﬁdence intervals.
Unﬁlled circles represent raw data and for clarity are offset to the right when the

 

50-

40-

30-

20-

10-

20-

15-

10-

 

 

 

 

 

 

 

- Top 128 cell
[:1 Mid 288 cell

 

1 28/288

same value is repeated.

 

E

 

 

 

 

 

 

 

C
O
O
F
O
- Top 288 cell - Side 288 cell
ems:- Mid 288 cell - Mid 288 cell
I: Bot 233 cell 8.»:- Side 288 cell
O
o
O
O é
288/288l288 288\ /288/288
Treatments

51

Total root plugs

APPENDIX

EFFECTS OF PHOTOPERIOD TRANSFERS AND COLD TREATMENTS ON
THREE ASTERACEAE SPECIES, COREOPSIS GRANDIFLORA ‘EARLY
SUNRISE’, LEUCANTHEMUM XSUPERBUM ’SNOW LADY’ AND
RUDBECKIA FULGIDA ’GOLDSTURM’.

52

Effects of Photoperiod Transfers and Cold Treatments on Three Asteraceae
Species, Coreopsis grandiﬂora ‘Early Sunrise’, Leucanthemum xsuperbum ‘Snow

Lady‘ and Rudbeckia fulgida ‘Goldsturrn’.

Joaquin A. Chong’, Royal Heinsz, Arthur C. Cameronz, and William Carisonz.

Department of Horticulture, Michigan State University, East Lansing, MI 48824-1325

Received for publication . Accepted for publication . We
gratefully acknowledge the ﬁnancial support of the Agricultural Experiment
Station of Michigan State University and funding by producers and greenhouse
growers supportive of Michigan State University ﬂoricultural research.

’ Graduate Student.

2 Professor.

53

Abstract

The effects of photoperiod transfers between short and long days or cold
treatment on ﬂowering, height, and bud number of three Asteraceae plants,
Coreopsis grandiﬂora Hogg ex. Sweet “Early Sunrise‘, Leucanthemum
xsuperbum L. “Snow Lady’, and Rudbeckia fulgida Ait. ‘Goldstunn’ were
determined. Plants were grown to maturity under 9- (SD) or 16-h (LD)
photoperiods, and thereafter were treated with an opposite photoperiod (SD or
LD) duration or a cold period of 0, 1, 2, 3, 4, 5, 6, 8, or 10 weeks. After
completion of the opposite photoperiod duration, plants were transferred to their
initial photoperiod or placed under a 16-h photoperiod after cold. To ﬂower,
plants did not require SD or cold before LD. All plants under continual LD
ﬂowered faster than either SD- or cold-treated plants, with the exception Of
Rudbeckia, which ﬂowered the fastest with 6 weeks of LD intercalated between
SD. Rudbeckia exhibited abnormal lateral ﬂowering. Rudbeckia plants of the
SD-LD-SD treatment or SD before cold had less abnormal ﬂowering than plants
under LD-SD-LD treatment or L0 before cold. Bud number and plant height on
Coreopsis and Rudbeckia increased with increasing LD and decreased with
increasing SD. In Leucanthemum, SD-LD-SD treatments did not affect plant

height or ﬂower bud number.

Introduction

For growers to successfully force herbaceous perennials into ﬂower for
speciﬁc market dates, speciﬁc photoperiodic and cold requirements as well as
the cultural information to control plant height and architecture are required.
Species of Asteraceae such as Coreopsis grandiﬂora ‘Early Sunrise’,
Leucanthemum xsuperbum ‘Snow Lady’, and Rudbeckia fulgida ‘Goldsturm’ are
popular perennials, ranking among the top 10 best-selling species (Rhodus,
1995). Plant architectural changes could allow many new marketing
opportunities, since plant height speciﬁcations are important to meet shipping
requirements.

In 1996, C. grandiﬂora ‘Early Sunrise’ and L. xsuperbum ‘Snow Lady‘ were
seeded in early July and grown for sale in late summer in a commercial
greenhouse. Most plants in the population failed to set ﬂower buds and had to
be discarded. Throughout the crop’s growth, the growing environment was
characterized by high temperatures and a photoperiod of continual natural long
days (LD). Traditionally C. grandiﬂora ‘Early Sunrise’ and L. xsuperbum ‘Snow
Lady’ species are seeded under natural short days (SD) and then transferred to
L0 for ﬂowering. (The failed crop was never exposed to SD because of its
seeding date. Summer temperatures during 1996 were very high in Michigan.
Failure to ﬂower could have been due to Coreopsis and Leucanthemum’s having
the dual photoperiod requirement of SD followed by LD; alternatively, ﬂowering

could have been delayed and inhibited by high summer temperatures.

55

Three main photoperiodic categories for ﬂowering in plants are common,
short day, long day, and day indifferent or day neutral. Less common
photoperiodic categories include intermediate-day plants and dual-daylength
plants. Intermediate-day plants ﬂower when the day is neither too short nor too
long (Brian and Vince-Pure, 1997). Dual-daylength plants require a two step
ﬂower induction process consisting in a primary induction photoperiod of either
SD or LD followed by a secondary induction photoperiod for ﬂowering (Blondon,
1972; Heide, 1984). These inductive steps represent plant adaptations
developed in distinct latitudinal ecotypes in response to seasonal changes
(Heide, 1995). Dual-photoperiodic plants have adapted to seasonal changes to
germinate, grow, overcome juvenility, ﬂower, bear seed and continue their life
cycle under speciﬁc advantageous environmental conditions.

There are two main subdivisions in dual-photoperiodic plants. Plants that
require an SD followed by an L0 are called short-long-day plants (SLDP), and
those that require an LD followed by a SD are called long-short-day plants
(LSDP). LSDP ﬂower in the secondary induction, SD, after having had LD
exposure for primary induction. SLDP ﬂower in the secondary induction, LD,
after having had SD for primary induction. A dual-photoperiod plant exposed to
either continual L0 or SD should fail to initiate ﬂower. Photoperiodic categories
are not ﬁxed, but are modiﬁable by environmental conditions such as
temperature, light irradance and age (Brian and Vince-Prue, 1997).

Some species of Asteraceae such as C. grandiﬂora, which are native from

Arizona (lat. 32° N, long. 111° W) to Minnesota (lat. 44° N, long. 95° W), might

56

require a dual photoperiod to ﬂower. Ketellapper and Barbaro (1966) found C.
grandiﬂora Nutt. offspring of var. Single Mayﬁeld Giant required an SD before LD
for ﬂowering induction and that the SDcould be replaced by low temperatures.
Experiments with L. vulgare Lam. var. Oxeye daisy grown from seeds collected
in northern Norway showed that the plants required dual photoperiodic induction
for ﬂowering (Heide, 1995).

As mentioned, photoperiodic categories are not ﬁxed, but modiﬁable.
Requirements for vemalization can sometimes be substituted partially or entirely
by SD photoperiods (Evans, 1987; Heide, 1986 and 1995). Plants whose SD
inductive treatments can substitute for vemalization treatments can be classiﬁed
as SLDP or as a cold-requiring LDP (Heide, 1995; Brian and Vince-Prue, 1997).
Evans (1987) showed low temperature vemalization could be replaced entirely
by SD in some European winter wheat varieties, especially Templar.
Unvemalized SD-treated wheat varieties Templar and Huntsman had faster
inﬂorescence initiation than vemalized plants during secondary inductive LD
(Evans, 1987). Plants whose SD cannot replace vemalization requirements are
consider not SLDP but vernalization-requiring LD plants (Brian and Vince-Prue,
1997). ‘Oxeye’ daisy is such a plant (Heide, 1995).

Long day vemalization is also possible and is referred to as the
substitution of cold requirement with LD. Long day vemalization occurs in
Scabiosa columbaria L., and Campanula Iongistyla Fomin. (Mathon, 1960). Little

is known about the mechanism of LD vemalization.

57

Requirements of cold and photoperiod and the effects these have on
ﬂowering and plant height are pertinent the perennial greenhouse industry. The
goal of our research was to deﬁne whether three species from Asteraceae, C.
grandiﬂora ‘Early Sunrise‘, L. xsuperbum ‘Snow Lady’ and R. fulgida ‘Goldsturm’,
required an SD or cold treatment before LD for rapid and uniform ﬂowering and

to assess the effects of these treatments on plant height.

58

Materials and methods

Plant material

Experiments were conducted in the Plant and Soil Science Research
Greenhouses at Michigan State University, East Lansing, Michigan.

Seed propagated C. grandiﬂora “Early Sunrise’, L. xsuperbum ‘Snow Lady’
and R. fulgida ‘Goldsturm’ arrived from a commercial greenhouse (C. Raker &
Sons, Inc., Litchﬁeld, Mich.) on January 17, 1998, in size 288 plug trays (6 ml per
cell). Plant material had two false leaves (cotyledons) and zero to two nodes.
Plants for replicates were at the same stage of development when received on
March 1, 1998.

Initial Photoperiod Treatments

Plug trays were placed under a 9- or 16-h photoperiod upon arrival and
were singulated, leaving a single plant per cell. Plants under the 16-h
photoperiod were lighted from 0800 to 2400 HR with 400-W high-pressure
sodium (HPS) lamps. Plants under 9-h photoperiods were covered with opaque
black cloth from 1700 until 0800 HR. Photoperiod (1700 to 2400 HR) and
supplemental (0800 to 1700 HR) lighting were switched on during the indicated

part of the day if the ambient greenhouse photosynthtic photon ﬂux (PPF) was
below 200 meI'm‘2'8'1 and switched off if it exceeded 400 umol-m'Z-S'I. The
HPS lamps provided a minimum PPF of 50 umol-m'Z-S'1 and an average of 65
umol-m'Z-S"1 at plant height.

Plants were transplanted to size 32 plug trays, 130 ml per cell (Summit

Plastic Co. Tallmadge, Ohio, after approximately two weeks. Trays were ﬁlled

59

with a commercial soilless medium composed of pine bark, vermiculite, Canadian
sphagnum peat, and coarse perlite with a wetting agent, lime, and starter
fertilizer charge (High Porosity Mix, Strong-Lite Products, Pine Bluff, Ark).

Photoperiod transfers and cold treatments

Coreopsis grandiﬂora plants bulked under the 9- or 16-h photoperiod had
developed ~8 nodes by March 17, 1998, and R. fulgida had developed ~10 by
March 30, 1998. Photoperiod transfers and cold treatments began on these
dates. Leucanthemum xsuperbum plants under 9-h photoperiod reached ~13
nodes by March 17, 1998, and were treated with cold and photoperiod transfers
for the same duration as C. grandiﬂora and R. fulgida. However, L. xsuperbum
plants under 16-h photoperiods induced ﬂowers, and all plants developed visible
ﬂower buds; therefore, they were not treated with cold or photoperiod transfer.

In the replicate experiment, C. grandiﬂora bulked under the 16-h

photoperiod had developed ~8 nodes by April 20, 1998, and those under the 9-h
photoperiod developed ~8 nodes by April 29, 1998. Rudbeckia fulgida in both
photoperiods reached ~10 nodes by May 13, 1998, and L. xsuperbum in 9-h
photoperiods reached ~13 nodes by April 23, 1998. All L. xsuperbum plants
under 16-h photoperiods induced ﬂowers and were not treated with cold or
photoperiod transfer.

Photoperiod transfer

Photoperiod-treated plants were transplanted from size 32-cell plugs to
13-cm square plastic containers (1.1 L) with the same medium the cold-treated

plants had. Eighty plants bulked under 9-h photoperiods were transferred to a

60

16-h photoperiod for 1, 2, 3, 4, 5, 6, 8, or 10 weeks, and 10 plants were kept
under 9-h photoperiods as a control (Fig. 1). After each 16-h photoperiod
duration, plants were returned to the 9-h photoperiod. Similariy, 80 plants bulked
under 16-h photoperiods were transferred to a 9-h photoperiod for 1, 2, 3, 4, 5, 6,
8, or 10 weeks, and 10 plants were kept under the 16-h photoperiod as a control.
After each 9-h photoperiod duration, plants were returned to the 16-h
photoperiod.

In the replicate experiment, plants under the 16-h photoperiod were
transferred to 9-h photoperiods for 1, 2, 3, 4, or 5 weeks and plants under 9-h
photoperiods were transferred to 16-h photoperiods for 1, 2, 3, 4, or 5 weeks.
Ten plants were left under each initial photoperiod treatment as controls.

Cold treatments

Cold consisted of either no cold or transfer of plants to a 5 °C controlled-
environment chamber for 1, 2, 3, 4, 5, 6, 8, or 10 weeks. In the replicate
experiment, cold treatments were no cold or 1, 2, 3, 4, or 5 weeks. The
controlled environment chamber was divided, and plants were exposed to a
photoperiod of 9 or 16 h. Lighting was from cool-white ﬂuorescent
(F96T12/CWNHO, Philips, Somerset, NJ.) lamps from 0800 to 1700 HR for both
photoperiods. Incandescent lamps completed the 16-h photoperiods as a day

extension from 1700 to 2400 HR. The PPF from the cool-white ﬂuorescent lamps
was ~100 pmol-m'Z-S'I; from the incandescent lamps ~1 to 3 umol-m'Z-S'I.

Plants in the chamber were watered as needed (approximately two times per

61

week) with well water acidiﬁed with sulfuric acid (H2804) to a pH of

approximately 6.0.

Plant node counts were made after cold treatments, and plants were
transplanted to 13-cm square terracotta plastic containers (1.1 L) ﬁlled with the
same medium (High Porosity Mix, Strong-Lite Products, Pine Bluff, Ark.) After
transplant, plants were forced under a 16-h photoperiod with daylength extension
from 1700 to 2400 HR with HPS lamps, as described in the initial photoperiod
protocol. Natural daylength was supplemented from 0800 to 1700 HR. as
previously discussed. Settings and PPF were as described for the initial
photoperiod treatments.

Plant culture

Plants were top-watered and fertilized at every irrigation throughout all
experiments. The nutrient solution was of well water (EC Of 0.70 mS cm'1 and
105, 35, and 23 [mg L'I] Ca, Mg, and S, respectively; pH 6.0) acidiﬁed with

H2804 to a titratable alkalinity of 130 mg CaCO3 L'1 and water soluble fertilizer

providing 125-12-125-13 N-P-K-Ca (mg L4; 30% ammoniacal N) plus 1.0, 0.5,
0.5, 0.5, 0.1, 0.1 (Fe, Mn, Zn, Cu, B, MD; mg L'I; MSU Special, Greencare
Fertilizers, Chicago, Ill.

Temperature Settings

Plants in the ﬁrst experiment were bulked at an average daily temperature
(ADT) of 23 °C for three weeks and 20 °C thereafter. Plants of the repetition
experiment were bulked at a constant 20 °C ADT from the beginning of the

experiment. Average daily temperature and daily light integral were monitored

62

with a CR-10 datalogger (Campbell Scientiﬁc, Logan, Utah) using 36-gauge type
E thermocouples and quantum sensors (Ll-COR), respectively. The datalogger
collected temperature and light measurements every 10 and recorded the hourly
average. Under each black cloth, heat was provided when needed to maintain a
night temperature of 20 °C by using a 1500-W electric heater (Model T771, Rival
Manufacturing Co, Sedalia, Mo.)

Data Collection

In the cold experiment, node counts were made after the end of each cold
treatment. In the photoperiod transfer experiment, node counts were made when
plants were transferred from their bulking photoperiod to the 9- or 16-h
photoperiod. All plant nodes were counted, and the ﬁnal leaf node produced was
marked with Liquid Paper correction ﬂuid (‘Bond White‘; Gillette 00., Boston,
Mass.) At anthesis ﬁnal node count was taken on all plants from the marked leaf
to the ﬂower or ﬂower shoot.

Days to visible bud and ﬂower were calculated on all reproductive plants.
In the cold experiment, calculations were made from the seeding date and from
the day cold duration ended. In the photoperiod transfer experiment, days to
visible bud and days to ﬂower were calculated from seeding. Flowering
percentages were calculated by dividing the number of plants that ﬂowered by
the plants that were forced. Plant height was recorded on all three species.

The cold duration experiment was analyzed as a factorial design with
three factors, initial photoperiod, cold duration and photoperiod during cold. The

photoperiod transfer experiment was analyzed as a factorial with two factors,

63

initial photoperiod and transfer duration. Ten plants per treatment were used in
all experiments, except for L. xsuperbum which had eight plants per treatment
during the ﬁrst experiment.

Initial and ﬁnal node number; days to visible bud, ﬂower, and from visible
bud to ﬂower; plant height; and ﬂower bud number data were analyzed with
SAS’s (SAS Institute, Cary, NC) analysis of variance (ANOVA) and a general

linear model (GLM) procedure.

64

Results

Dual-photoperiod experiment

Coreopsis grandiﬂora. All plants bulked under LD (10 weeks) from
seeding ﬂowered, irrespective of SD treatment duration (Fig. 2A). However, time
to visible bud and ﬂower increased as SD duration increased. Plants bulked
under SD from seeding required only two weeks of LD for all plants to ﬂower (Fig.
23); 80% of plants grown under continual SD ﬂowered, but ﬂowering was
delayed about 50 days compared with that of grown under continual LD. Days to
visible bud and ﬂower decreased as LD duration increased for SD-LD-SD plants.
Flower bud number (Fig. 20) and plant height (Fig. 2D) generally increased as
LD duration increased in SD-LD-SD plants. In contrast, ﬂower bud number and
plant height decreased as SD duration increased in LD-SD-LD plants. Total leaf
number differed signiﬁcantly between initial photoperiod treatments but was not
statistically signiﬁcant for weeks of transfer photoperiod (data not shown).

Leucanthemum xsuperbum. All plants bulked under LD initiated ﬂowers
after forming approximately 20 nodes and ﬂowered before SD treatment; thus, no
data are reported. All SD-bulked plants ﬂowered under SD following only 1 week
of LD (Fig. 3A). Days to visible bud and ﬂower decreased as LD duration
increased from 0 to 2 weeks; thereafter, they remained the same. Plant height
(Fig. 3B) and ﬂower bud number (Fig. BC) increased as LD duration increased
from 0 to 5 weeks by 7.5 cm and 19 buds, respectively.

Rudbeckia fulgida. All plants bulked under continual LD ﬂowered (Fig.

4A). All LD-bulked plants exposed to 8 or fewer weeks of SD ﬂowered; only 60%

65

of plants ﬂowered when exposed to 10 weeks of SD following LD bulking. Days
to visible bud and ﬂower increased as SD duration increased up to 6 weeks for
LD-SD-LD plants. Further increases in SD duration did not increase days to
visible bud or ﬂower. Flower bud number (Fig. 40) increased on LD-SD-LD
plants as SD increased to 4 weeks; thereafter, ﬂower bud number decreased,
especially between 6 and 8 weeks of SD. Plants bulked under SD required at
least 4 weeks of LD for all plants to ﬂower (Fig. 48). Days to visible bud and
ﬂower decreased as LD duration increased to 6 weeks. Flower bud number (Fig.
4C) and plant height (Fig. 4D) generally increased with LD duration increase in
SD-LD-SD plants. Node counts were not statistically signiﬁcant between initial
photoperiods and weeks of transfer photoperiod (data not shown).

Cold treatment experiment

Coreopsis grandiﬂora. All plants ﬂowered irrespective of cold treatment
(data not shown). Photoperiod during cold treatment had no signiﬁcant effect on
time to ﬂower; therefore, data from both photoperiods were pooled for further
analysis. The time from the end of cold treatment to ﬂower progressively
decreased as cold duration increased (Fig. 5A). However, when time to ﬂower
was calculated from seeding, total time increased as cold duration increased.
Plants bulked under LD before cold ﬂowered up to 6 days faster than those
bulked under SD (P 5 0.0001). Flowering was relatively homogeneous within
treatments, as shown by very tight 95% conﬁdence intervals. Plant height at
ﬂower increased about 10 cm as cold duration increased from 0 to 6 weeks (Fig.

SB); photoperiod before cooling had no effect on ﬂowering height. Flower

66

number increased 38 or 41% as cold duration increased from 0 to 3 weeks or 0
to 4 weeks for SD and LD pretreated plants, respectively (Fig. 50). Further
increases in cold duration did not increase bud number. Total node counts were
insigniﬁcant between initial photoperiods but signiﬁcant with cold treatment.
Total node count generally decreased quadratically (P 5 0.0001) and linearly (P 5
0.0008) with increasing in cold, suggesting earlier induction caused by cold
treatment.

Leucanthemum xsuperbum. All plants bulked under LD before cold
exhibited ﬂower buds before cold; thus, only days to visible bud and ﬂower are
reported (Fig. 6A). All SD bulked plants ﬂowered, irrespective of cold treatment
duration (data not shown). Time to ﬂower following cold treatment decreased 13
days as cold duration increased to 10 weeks for SD-bulked plants (Fig. 6A).
However, total time to ﬂower from seeding increased 57 days as cold duration
increased from 0 to 10 weeks. Time to ﬂower was signiﬁcantly affected by
photoperiod during the cold treatment. However, since the cold-treated plants
under LD ﬂowered only 1 to 2 days faster than plants under SD, data were
pooled: there was no statistical interaction, and the 1- to 2-days difference was
judged horticulturally insigniﬁcant. There was no consistent trend between cold
duration and plant height or ﬂower bud number (Fig. 6B and Fig. 60). Plant
height averaged 24 cm and ﬂower bud number averaged 26 buds across all cold
durations. Total node number had a linear trend (P 5 0.001).

Rudbeckia fulgida. All plants ﬂowered, irrespective of cold treatment (data

not shown). Photoperiod during cold treatment, as with Coreopsis, was

67

insigniﬁcant, and data were pooled for further analysis. The time to ﬂower from
the end of cold treatment progressively decreased by up to 29 days as cold
duration increased (Fig. 7A). However, when calculated from seeding, total time
to ﬂower increased by up to 39 days. Plants bulked under SD before cold
ﬂowered up to 11 days faster than those bulked under LD (P 5 0.0001). Both
plant populations had homogeneous ﬂowering within treatments, as shown by
tight 95% conﬁdence intervals. The percentage of plants ﬂowering from a lateral
shoot generally decreased as cold duration increased; ﬂowering from lateral
shoots was also less prominent in plants bulked under SD before cold (Fig. 78).
Plants bulked under LD before cold tended to have more ﬂower buds than plants
bulked under SD for cold durations shorter than 8 weeks (Fig. 7C). Plant height
at ﬂower was similar on plants from all cold treatments, and average height was
51 cm (Fig. 70). Total node number was signiﬁcant (P 5 0.002) between initial
photoperiods and decreased quadratically and linearly for 9-h photoperiods and

linearly for 16-h photoperiods (data not shown).

68

Discussion

The main purpose of this experiment was to determine whether SD or cold
was required before LD for rapid uniform ﬂowering of C. grandiﬂora ‘Early
Sunrise‘ and L. xsuperbum ‘Snow Lady’. The experiment was initiated because
during the summer of 1996, plants of these species grown under natural
continual LD and high temperature in a commercial greenhouse failed to ﬂower
uniformly. In our experiment all C. grandiﬂora, L. xsuperbum and R. fulgida,
grown under continual LD ﬂowered with or without cold. The data showed that
neither SD nor cold was required before LD for ﬂower induction of these species,
disproving our hypothesis. However cold treatment decreased time to ﬂower
from start of inductive LD in all species tested (Fig. 5A, 6A, and 7A). As Harkess
and Lyons (1994) found with Rudbeckia, our Coreopsis and Rudbeckia plants
ﬂowered when transferred to SD following LD induction, and plants were shorter
compared with those grown under continual LD.

Based on these results, it seems more probable that plants grown during
the summer of 1996 were exposed to supra-optimal temperatures, causing
ﬂowering delay and improper plant growth. In a preliminary experiment, we
found that plants grown . under continual 35°C demonstrated similar
characteristics: some failed to ﬂower, and all grew abnormally.

The most rapid ﬂowering C. grandiﬂora and L. xsuperbum occurred under
continal LD without cold or SD. However, R. fulgida ﬂowered fastest under the
SD-LD-SD treatment consisting in 6 weeks of LD intercalated between SD. The

plants ﬂowered 12 to 14 days earlier than those in continual LD or LD-SD-LD,

69

respectively (Fig. 4 and 7). The data suggest that R. fulgida 'Goldsturm' is the
only species in these experiments that beneﬁts from SD before LD treatment for
most rapid ﬂowering.

Plant architecture and height can be controlled with photoperiod
treatments (Damann and Lyons, 1993). Rudbeckia and Coreopsis plants bulked
under 9-h photoperiods had a smaller leaf area than those bulked under 16-h
photoperiods. As plants bulked under SD were transferred to LD new developed
leaves increase its size. Conversely, new developed leaves of plants bulked
under LD decreased in size when plants were transferred to SD. Rudbeckia had
abnormal lateral-shoot ﬂowering, which meant the apical meristem failed to
develop or development was slower than that of lateral shoots, which reached
anthesis ﬁrst. In general, SD pretreated Rudbeckia plants had less abnormal
ﬂowering than LD pretreated plants. In the photoperiod-transfer experiment, SD-
LD-SD plants had ~28% abnormal ﬂowering compared to ~54% abnormal
ﬂowering in LD-SD-LD plants averaged across all treatments (data not shown).
Similarly, plants bulked under SD before cold had less abnormal ﬂowering than
plants bulked under LD before cold (Fig. 73).

Although SD and cold are not required before LD for ﬂowering, and
photoperiods during cold treatment had no horticulturally signiﬁcant effect on time
to ﬂower, treatment combinations of SD and LD duration changed plant
morphology and architecture. Effects were greatest on C. grandiﬂora and R.

fulgida whose plant height and bud number, both horticulturally important

70

variables, were affected. Generally plant height and bud number increased with
increasing LD duration and decreased with increasing SD duration.

Damann and Lyons (1993) expanded on Mumeek’s (1936) concept of
“photoperiodic inhibition,” deﬁning limited inductive photoperiod (LIP) as a plant‘s
being given a minimum number of LD inductive cycles to initiate ﬂowering and
then being transferred to noninductive conditions. Our data are similar to those
from studies of Damann and Lyons (1993), where an increase in inductive LD
increased plant height of two Coreopsis species.

In L. xsuperbum, SD-LD-SD treatments did not affect plant height or
ﬂower bud number; however, LD intercalated between SD increased C.
grandiﬂora and R. fulgida plant height and ﬂower bud number and decreased
days to visible bud and ﬂower with increasing LD duration. The reverse of SD-
LD-SD were LD-SD-LD treatments, or what we call limited noninductive
photoperiods (NIP). Noninductive SD photoperiod treatments were intercalated
between LD inductive photoperiods. Our data, like that of Harkess and Lyons
(1994) in their publication on Rudbeckia hirta, show that increasing SD durations
generally decreased Rudbeckia and Coreopsis plant height and bud number and
increased days to visible bud and ﬂower. In C. grandiﬂora and R. fulgida, LD-
SD-LD plants (NIP) had greater mass than SD-LD-SD plants (LIP), which were
generally smaller.

The LIP and NIP methods Of photoperiod control are potentially important
for the perennial industry, since they provide nonchemical height-control methods

capable of changing plant architecture, especially height.

71

In summary, C. grandiﬂora ‘Early Sunrise”, L. xsuperbum ‘Snow Lady’, and
R. fulgida ‘Goldsturm’ did not require an SD or cold before LD for ﬂowering.
However, SD or cold before L0 in Rudbeckia and a cold treatment in
Leucanthemum and Coreopsis decreased time to ﬂower following the cold

treatment.

72

References

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glomerata L. In: P. Chourard [et] and N. de Bilderling, eds. Phytotronique
et Prospective Horticole. Gauthier-Villards, Paris.

Brian, T. and Vince-Prue, D. 1997. Photoperiodism in plants. Academic Press,
San Diego.

Damann, MP. and Lyons, RE. 1993. Juvenility, ﬂowering, and the effects of a
limited inductive photoperiod in Coreopsis grandiﬂora and C. Lanceolata.
J. Amer. Soc. Hort. Sci. 1 18(4)2513-518.

Evans, LT. 1987. Short day induction of inﬂorescence initiation in some winter
wheat varieties. Aust. J. Plant Physiol. 14:277-286.

Harkess, R.L. and Lyons, RE. 1994. Floral initiation in Rudbeckia hirta
(Asteraceae) under limited inductive photoperiodic treatments. Amer. J.
Bot. 81 (8)21021-1026.

Heide, OM. 1984. Flowering requirements in Brpmus inennis, a short-long-day
plant. Physiol. Plant. 62:59-64.

Heide, OM. 1986. Primary and secondary induction requirements for ﬂowering
in Alopecums pratensis. Physiol. Plant. 66:251-256.

Heide, OM. 1995. Dual induction control of ﬂowering in Leucanthemum vulgare.
Physiol. Plant. 95:159-165.

Ketellapper, H.J. and Barbaro, A. 1966. The role of photoperiod, vemalization
and gibberellic acid in ﬂoral induction in Coreopsis grandiﬂora Nutt. Phyton
23(1):33-41.

Mathon, C.—Ch. 1960. Temperature de vemalisation et exigences
photopériodiques. Société Botanique de France. 107292-93.

Mumeek, A.E. 1936. A separation of certain types of responses of plants to
photoperiod. Proc. Amer. Soc. Hort. Sci. 34:507-509.

Rhodus, T. 1995. Top 20 Perennials. Greenhouse Grower Jan.: 80-82.

73

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Weeks of SD or LD

Figure 1. Schematic representation of photoperiod transfer experiment. Black
ﬁlled bars represent initial photoperiod under 9- (SD) or 16-h (LD) photoperiods
and gray bars represent the transfer photoperiod duration. Plants were under the
initial photoperiod for 10, 11, or 13 weeks for Coreopsis, Leucanthemum, and
Rudbeckia, respectively. After the initial photoperiod treatment, plants were
moved to the opposite photoperiod for 0, 1, 2, 3, 4, 5, 6, 8, or 10 weeks and then
transferred back to the initial photoperiod if anthesis was not reached.

74

Plant height (cm) at anthesis

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Flowering percentage (

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

    

 

 

 

 

 

 

 

 

 

 

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75

 

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Flower bud number
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Weeks of LD

Figure 3. The effects of SD-LD-SD (SD = short days; LD = long days) on days to
visible bud, days to ﬂower, ﬂowering percentage (A), plant height (B), and ﬂower
bud number (C) of Leucanthemum xsuperbum ‘Snow Lady’. L = linear; Q =

quadratic trends. NS,*,*** nonsigniﬁcant or signiﬁcant at P 5 0.05 or 0.001,
respectively.

76

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circles represent LD-SD-LD (NIP) treatment and black-ﬁlled circles represent SD-

‘ﬁcant at P < 0.05

long days) and SD-
nonsigniﬁcant or signi

ﬂowering percentage, ﬂower

short days; LD
and plant height (D) of Rudbeckia fulgida ‘Goldsturm’.

77

9

quadratic trends. NS

linear; Q =
or 0.001, respectively.

LD-SD (LIP) treatments in (C) and (D). Error bars are 95% conﬁdence intervals.

LD-SD (B) on days to visible bud, days to ﬂower

Figure 4. The effects of LD-SD-LD (A; SD
bud number (C)

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A

 

    
 

 

 

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Plant height (cm)
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Flower bud number

 

 

 

 

0 2 4 6 8 10
Weeksat5°C

Figure 5. The effects of cold treatment on days to ﬂower (A), plant height (B),
and ﬂower bud number (C) of Coreopsis grandiﬂora ‘Early Sunrise’. Days to
ﬂower were calculated from seeding and the end of cold. Black-ﬁlled symbols
represent SD before cold; unﬁlled symbols, LD before cold. Error bars are 95%
conﬁdence intervals. L = linear; Q = quadratic trends. NS,*,*** nonsigniﬁcant or
signiﬁcant at P 5 0.05 or 0.001, respectively.

78

Days to ﬂower

Flower bud number

Plant height (cm)
at ﬂower

160

120 -

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40

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Weeks at 5°C

Figure 6. The effects of cold treatment on days to ﬂower (A), plant height (B) and
ﬂower bud number (C) of Leucanthemum xsuperbum ‘Snow Lady’.
ﬂower were calculated from seeding and the end of cold. Black-ﬁlled symbols
represent SD before cold. Error bars are 95% conﬁdence intervals. L = linear; Q
= quadratic trends. NS,*,**,*** nonsigniﬁcant or signiﬁcant at P 5 0.05, 0.01, or
0.001, respectively.

79

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Lateral ﬂowering percentage Plant hei

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quadratic trends.

, 0.01, or 0.001, respectively.

linear; Q

L
< 0.05

Days to ﬂower were calculated from seeding and the end of cold.

Black-ﬁlled symbols represent SD before cold; unﬁlled symbols, LD before cold.

Error bars are 95% conﬁdence intervals.
nonsigniﬁcant or signiﬁcant at P

'k ** ***

Figure 7. The effects of cold on days to ﬂower (A), lateral ﬂowering percentage
(B), ﬂower bud number (C), and plant height (D) of Rudbeckia fulgida

‘Goldsturm’.
NS

80