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dissertation entitled

FACTORS INFLUENCING PHENOTYPIC VARIABILITY
IN MICROPROPAGATED STRAWBERRY
(FRAGARIA x ANANASSA DUCH.) CULTIVARS

 

presented by

J. Scott Cameron

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in Horticulture

 

 

 

I Major professor

Date _‘_’3_°[fifi

 

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

 

 

 

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FACTORS INFLUENCING PHENOTYPIC VARIABILITY
IN NICROPROPAGATED STRAWBERRY
(FRAGARIAXANANASSA DUCH.) CULTIVARS

 

BY

J. Scott Cameron

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Horticulture

.1986

ABSTRACT

FACTORS INFLUENCING PHENOTYPIC
VARIABILITY IN MICROPROPAGATED STRAWBERRY
(FRAGARIAXANANASSA DUCH.) CULTIVARS

 

BY

J. Scott Cameron

The field performances of progeny of micropropagated
(MP) and conventionally propagated (CP) plants of
Junebearing strawberry cultivars were compared in matted
rows. Yields from MP rows were significantly greater for
three of six cultivars; this was primarily due to higher
plant density.

In a greenhouse study, detached primary, secondary,
and tertiary runner plants of MP 'Redchief' and 'Earliglow'
responded differently than CP plants of similar age. Total
flower number was significantly greater in all asexual
progeny of MP 'Redchief' plants due to increases in number
of trusses and flowers per truss. Fruit size was reduced
in all progeny generations of MP fRedchief'. Anthesis
was earlier in all progeny of MP 'Earliglow', and these
plants had significantly greater numbers of stolons.
Reduced numbers of flower trusses per plant and flowers
per truss were noted in tertiary progeny of MP 'Earliglow'.

In the field, progeny of MP 'Redchief' and
'Earliglow' had higher plant densities and yields than CP
plants, but partitioned 9-10 percent less of their total
dry matter to leaf tissue. Leaf area per crown was

significantly less in MP 'Redchief' (55 percent) and

'Earliglow' (49 percent). Yield per crown was significantly
reduced in MP 'Redchief' (32 percent) but not in
'Earliglow'. Net photosynthetic (PN) rate and stomatal
conductance (gs) was greater in MP 'Earliglow' but not
'Redchief'. Mesophyll conductance (gm) was unchanged in
both cultivars.

Gas exchange behavior in relationship to increasing
leaf density (leaf number per 0.093 m2) was compared in
matted rows of MP and CP 'Earliglow' and 'Redchief'.

Highly significant (p < 0.001) negative linear correlations
were observed between weight per leaf unit area and leaf
density. Significant, negative linear correlations were
also observed between PN, gs, gm and leaf density in

GP cultivars but not in MP plants. In a study examining
the influence of shading on MP plants of 'Redchief' and
'Earliglow' and their first and second year field grown
asexual progeny, no differential effect of shading was
noted. PN, gs, gm and chlorophyll content declined with
successive generations of conventional propagation.

Quantum yield as well as carboxylation efficiency and

gm response to increasing ambient C02 levels was greater in
MP than CP 'Earliglow'; these differences were not observed

in 'Redchief'.

Dedicated with love to
Nancy, Matthew and Evan

iv

ACKNOWLEDGMENTS

I wish to express my sincerest appreciation to
Dr. J. F. Hancock, my major professor, for his support and
encouragement during my graduate studies. His advice,
patience and sense of humor were assets to my program. I
would also like to thank Dr. J. A. Flore for his advice and
for allowing me to use his equipment and facilities for
gas exchange measurements.

I also wish to thank my wife, Nancy, for her love and
patience during my graduate years. Her unwavering support
and God-given understanding has helped to make this degree

a reality.

Guidance Committee:

The journal paper format was chosen for this thesis in
accordance with departmental and university regulations.
The thesis is divided into four sections. Section one is
intended for publication in Recent Advances in Strawberry
Production. Section two is intended for publication in
HortScience. Section three is intended for publication
in Scientia Horticulturae. Section four is intended for
publication in The Journal of the American Society for

Horticultural Science.

vi

TABLE OF CONTENTS

PAGE

List of Tables . . . . . . . . . . . . . . . . . . . . viii

List of Figures . . . . . . . . . . . . . . . . . . . x
IntrOdUCtion O O O O O O O O O O O O O O O O O O O O O 1
Section
I. The Field Performance of Strawberry Nursery Stock
Produced Originally From Runners or
Micropropagation . . . . . . . . . . . . . . . . 11
Abstract 0 O O O O O O O O O O O O O O O O O 12
Results and Discussion . . . . . . . . . . . 14
Literature Cited . . . . . . . . . . . . . . 17

II. Enhanced Vigor in Vegetative Progeny of
Micropropagated Strawberry Plants . . . . . . . . 19
Abstract . . . . . . . . . . . . . . . . . . 20
Literature Cited . . . . . . . . . . . . . . 27

III. The Influence of Micropropagation on Yield
Components, Dry Matter Partitioning and Gas

Exchange Characteristics of Strawberry . . . . . 28
Abstract . . . . . . . . . . . . . . . . . . 29
Introduction . . . . . . . . . . . . . . . . 29
Materials and Method . . . . . . . . . . . 31
Results . . . . . . . . . . . . . . . . . . 32
Discussion . . . . . . . . . . . . . . . . . 36
References . . . . . . . . . . . . . . . . . 40

IV. The Influence of Leaf Density and Shading on
Gas Exchange Characteristics of Micropropagated

Strawberry Plants . . . . . . . . . . . . . . . . 43
Abstract . . . . . . . . . . . . . . . . . . 44
Materials and Methods . . . . . . . . . . . 46
Results . . . . . . . . . . . . . . . . . . 50
Discussion . . . . . . . . . . . . . . . . . 70
References . . . . . . . . . . . . . . . . . 73

vii

LIST OF TABLES

 

 

TABLE 1:15;
Introduction
1 Species which have shown variation among
calli (C) or regenerated plants (R) . . . . . 2
2 Phenotypic behavior of micropropagated

strawberry plants directly from culture
compared with that of conventionally
propagated plants . . . . . . . . . . . . . . 5

Section One

 

1 Field performance of six strawberry
cultivars derived from nursery stock
propagated conventionally and by
micropropagation . . . . . . . . . . . . . . . 15

Section Two

 

1 Sexual responses of detached primary (1°),
secondary (2°) and tertiary (3°) daughter
plants from standard (ST) and micropro-
pagated (MP) mother plants of strawberry.
Numbers in parentheses represent
difference (%) between MP and ST plants . . . 24

2 Vegetative response of detached primary
(1°), secondary (2°), and tertiary (3°)
daughter plant from standard (ST) and
micropropagated (MP) mother plants of
strawberry. Numbers in parentheses
represent difference (%) between MP and
ST plants . . . . . . . . . . . . . . . . . . 25

viii

Section Three

 

Yield components of plants in matted row
quadrats of micropropagated (MP) and
conventionally propagated (CP) 'Earliglow'
and 'Redchief' strawberry . . . . . . . . .

Biomass allocation in plant structures of
micropropagated (MP) and conventionally
propagated (CP) 'Earliglow' and 'Redchief'
strawberry . . . . . . . . . . . . . . . . .

Gas exchange, weight per leaf unit area,

and total chlorophyll in matted row quadrats
of micropropagated (MP) and conventionally
propagated (CP) 'Earliglow' and 'Redchief'
strawberry . . . . . . . . . . . . . . . . .

Section Four

 

Correlation coefficients of leaf density
(leaves per 0.093 m2) versus weight per leaf
unit area and gas exchange measurements in
matted rows of MP and CP 'Earliglow' and
'Redchief' strawberry . . . . . . . . . . .

Gas exchange characteristics of 'Earliglow'
and 'Redchief' strawberry plants of three
prppaggtion generations (PAR = 1200 umol

S m‘

) . . . . . . . . . . . . . . . . . .

Chlorophyll content of leaves from plants
of 'Earliglow' and 'Redchief' strawberry of
three propagation generations . . . . . . .

Characteristics of leaves from 'Earliglow'
and 'Redchief' plants of three propagation
generations grown under 100 percent and 21
percent full sun'. . . . . . . . . . . . . .

Maximum rates of net photosynthesis (PN) in
'Earliglow' and 'Redchief' strawberry plants
grown under different light levels.

Measurements made at PAR = 1200 umol S'1 m"2
and 26 1 1°C . . . . . . . . . . . . . . . .

ix

33

35

35

52

53

57

58

59

FIGURE

1

TABLE OF FIGURES

Affect of leaf density (number of

leaves per 0.093 m2) on net

photosynthesis (PN) on a weight per

leaf unit area basis in matted rows of
micropropagated ( ) and

conventionally propagated (----),
'Earliglow' (A) and 'Redchief' (B).

Each point represents a measurement of

one leaf with 21 replications per
propagation treatment . . . . . . . . . . .

 

Response of gas exchange parameters of
micropropagated (MP) (A, C, E) and
conventionally propagated (CP) (B, D,
F) 'Earliglow' plants grown under full
sun to different levels of
photosynthetically active radiation
(PAR). Gas exchange parameters: net
photosynthesis (PN) (A,B), mesophyll
conductance (gm) (C, D) and stomatal
conductance (gs) (E, F). Different
symbols represent replications; 6 - 10
measurements per replication . . . . . . . .

Response of gas exchange parameters of
micropropagated (MP) (A, C, E) and
conventionally propagated (CP) (B, D,
F) 'Redchief' plants grown under full
sun to different levels of
photosynthetically active radiation
(PAR). Gas exchange parameters: net
photosynthesis (PN) (A, B), mesophyll
conductance (gm) (C, D) and stomatal
conductance (95) (E, F). Different
symbols represent replications; 6 - 10
measurements per replication . . . . . . . .

PAGE

54

60

62

Response of net photosynthesis (mg CO

dm‘2 hr'l) to ambient C02 level in '
micropropagated (----) and

conventionally propagated ( )

'Earliglow' (A) and 'Redchief' (B)

potted plants grown under full sun . . . . . . 64

 

Response of net photosynthesis (ug C02
gm dr wt'1 hr'l) to ambient C02 level
in micropropagated (----), and
conventionally propagated ( )
'Earliglow' (A) and 'Redchief' (B)

potted plants grown under full sun . . . . . 66

 

Response of mesophyll conductance (gm)

on a weight per leaf unit area basis

to ambient C02 level in micropropagated

(~---) and conventionally propagated

( ) 'Earliglow' (A) and 'Redchief'

(B) potted plants grown under full

sun . . . . . . . . . . . . . . . . . . . . 68

 

xi

INTRODUCTION

The tissue culture environment can induce both
mutational and epigenetic changes that affect the growth
and development of plant cells, tissues and organs. While
these changes can provide a rich source of variability for
research and plant improvement (7,22), genetic mutations
can hinder the clonal micropropagation of selected
genotypes. Distinguishing between mutational and
epigenetically induced changes is often difficult, although
variations transmitted through meiosis are generally
considered to be genetic in origin (7).

Examples of in vitrg induced genetic and epigenetic
variability include cell cultures, callus cultures, and
regenerated plants (7,22,38). The variability generated
from these systems is thought to arise from several sources
including: 1) preexisting genetic differences in somatic
cells, 2) mutagenic activity of culture media or of
cellular constituents released by dying cells, 3)
chromosomal rearrangements, 4) gene amplifications and
deletions, and 5) cryptic virus elimination (38). Many
plant genera exhibit some form of variability as a result

of the in vitro environment (Table 1). These traits

 

appear to represent both genetic and epigenetic eVents, but
few of these systems have been fully characterized. While
the processes involved in dedifferentiation, cycles of
rapid cell division and redifferentiation introduce

l

2

Table 1. Species which have shown variation among
calli (C) or regenerated plants (R).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

7: y w s
4J-v-l U) B -r-l
gg g :2 8 >
pg; 8 g 5 §
e E1: .2 .. -
:3 851% 5 5-332 '°=’ a .5 :2
m -a u +1 u
” ‘52 “a '3 ‘3 m a 9 g
‘: ~ E g E ~ . ~
. 5% g E E a a
Allium oepa C C C C 35
Allium sativum R 32
Ananassa comosus R 47
Atropa belladonna C C C 11
Avena sativa R R 9
Brassica napus R 20
Brassica oleracea C R 17,20
Catharanthus roseus C 51
Citrus species C C 8,41
Diospyros kaki C 50
Haplopappus
gracilis C 43
Hedera helix C C C 1,29,42
Lactuca sativa R 37
Idthospermum
erythorrhizon C 30
Nicotiana tabacum C C C,R R C,R R R 12,27,28
‘ 35,40
Olea europea C 24
Oryza sativa R R 19,33
Pelargonium species R R R 39
Populus tremuloides C 49
Prunus serotina C C 6
Saccharum species R R R R 22,23,25
Solanum laciniatum C 52
Solanum tuberosum R R R. R 36,48
Sorghum bicolor R R 15
vaccinium
angustifolium C 31
vaccinium ashei C C 4,5
§§a_mays R 16

 

 

 

 

 

 

 

 

 

 

 

3

opportunities for genetic changes to occur in yi££g
(22,38), the mechanism(s) underlying phenotypic alterations
in clonal micropropagation systems are poorly understood.

Juvenility, or phase change, is a commonly cited
example of an epigenetic phenomenon and is most visible in
woody perennials (3). During the ontogeny of an
individual, expression of certain developmental and
morphological characters may change, including: ability to
flower, leaf cuticular characteristics, leaf shape and
thickness, phyllotaxis, thorniness, shoot orientation,
seasonal leaf retention, stem pigmentation, ability to form
adventitious roots and buds, patterns of photosynthate
partitioning, disease resistance, and cold resistance (18).

Clonal micropropagation appears to induce a: phase
change reversion in many woody plants. Mass
micropropagation systems which rely on axillary bud
proliferation induce physiological changes within the
initial explant which are analogous to juvenility (4,5,14).
In a number of species this often involves changes in
growth form and habit which precede rapid growth and
axillary bud proliferation. This "in vitro reversion" of a
mature explant does not appear to be complete, as resulting
plants do not display all tytdcal juvenile traits (18),
even though seedling morphology and behavior are often

induced in vitro (25). While epigenetic effects are often

4
minor and of short duration, the cultivated strawberry

(Fragaria x ananassa Duch.) may be an important exception.

Phenotypic Behavior of Micropropagated Strawberry Plants

A commercially-feasible method of strawberry
micropropagation was demonstrated by Boxus in 1974 (2).
Since that time, commercial laboratories in the United
States and Europe have produced millions of plants.
Several studies have shown that micropropagated plants
differ from their conventionally propagated counterparts in
many ways (Table 2), and that these differences may last
for at least the first growing season after culture
(10,34,44).

The cause(s) of phenotypic variability in
micropropagated strawberry cultivars is unknown. Genetic
mutation may play a role as several discrete mutants have
been identified (44); however, most populations of
micropropagated plants appear uniform with few deviants.
Phytohormones used in culture media may have post cultural
influences on morphology and physiology as these growth
regulators drastically alter the vegetative and
reproductive behaviors of the strawberry in 2239 (13,45)
and in vitrg (46). However, Marcotrigiano, §£.Elt (26)
reported that varying in vitrg levels of cytokinin
(benzylamino purine) did not alter subsequent stolon

formation in the field. Enhanced nutritional status could

Table 2: Phenotypic behavior of micropropagated
strawberry plants directly from culture compared with
that of conventionally propagated plants. Sources: (10,34,44).

 

Increased: Decreased:
vigor daughter plant crown size
runner plant production daughter plant leaf size
crown diameter crown diameter
crown production flowers per truss
stem diameter yield per crown
petiole length fruit weight
petiole/leaf length ratio disease resistance

number of flower trusses /plant
number of flower trusses /crown
flowers per plant

earliness of flowering
earliness of fruit ripening
yield per area

number of fruit per unit area
fruit production per plant

 

6
also play a role, but no information exists on the
elemental composition of micropropagated strawberries.

The strawberry is a unique test organism for
characterizing the nature of culture-induced variability.
It is perennial, prolific both sexually and asexually, and
is an economically important horticultural crop. However,
the long term affect of micropropagation beyond the first
year has not been explored, and the physiological basis of
enhanced vigor has not been elucidated.

The goals of this study were to: 1) determine if the
effects of micropropagation endure beyond the first year
and the mother plant derived from culture, and 2) determine
if micropropagation alters the yield components, gas
exchange properties and biomass allocation of Junebearing

strawberry cultivars.

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7

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8

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9

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10

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49. Winton, L.L. 1970. Shoot and tree production from aspen
tissue cultures. Amer. J. Bot. 57:904-909.

50. Yokoyama, T. and M. Takeuchi. 1976. Organ and plantlet
formation from callus in Japanese persimmon (Diospyros
kaki). Phytomorph. 26(3):273-275.

 

51. Zenk, M.H., H. El-Shagi, H. Arens, J. Stockigt, E.W.
Weiler and B. Deus. 1977. Formation of the indole alkaloids
sepentine and ajmaliacine in cell suspension cultures of
Catharanthus roseus. In:Plant Tissue Culture and its
Bio-technOIEgical Application (eds. W. Barz, E. Reinhard,
M.H. Zenk.), p. 27-43. Berlin:Springer.

 

52. Zenk, M.H. 1978. The impact of plant cell culture on
industry. In: Frontiers of Plant Tissue Culture 1978 (ed.
T.A. Thorpe), p. 1-13. Calgary:Int. Ass. Plant Tissue
Culture.

SECTION I

THE FIELD PERFORMANCE OF STRAWBERRY NURSERY STOCK PRODUCED
ORIGINALLY FROM RUNNERS OR MICROPROPAGATION

11

Abstract. The field performances of daughters of
micropropagated (tissue culture) and conventionally
propagated (runner) plants of Junebearing strawberry
cultivars were compared in matted rows. 'Yields from
micropropagated rows were significantly greater for three
of six cultivars. The primary factor for the improved
productivity was greater MP-derived plant density;
overcrowding may have reduced fruit weight in two
cultivars.

Field tests of micropropagated (tissue culture
produced) strawberry plants have demonstrated their
increased capacity for first-year vegetative growth and
runnering compared to standard, runner propagated plants
(3,7,8,9). Micropropagated plants have been suggested as a
superior source of stock for nursery production, especially
for shy-runnering cultivars (7,9). Previous studies have
examined plants directly from micropropagation and did not
test the performance of runner plants originating from
micropropagated nursery stock. Since one of the primary
factors limiting strawberry yields is matted row density
(5), planting stock derived from micropropagated plants may
provide a means for increasing plant numbers in the matted
row system. The following experiment assessed the
potential of micropropagation-derived plants of six

Junebearing cultivars for matted row culture in Michigan.

12

13
Materials and Methods

Nursery production. Indexed, virus-free plants were

 

obtained from the USDA Fruit Lab, Beltsville, Maryland, and
were planted in the spring of 1981 in screen houses in
South Deerfield, Massachusetts. These were allowed to
proliferate, and their daughters were used as standard
propagated plants (ST) and as stock plants for
micropropagation (MP). For micropropagation, plants were
moved to a greenhouse in the fall, and runner tips (5 mm
long) were removed. These explants were established on
standard Boxus (2) medium with no hormones. For
proliferation, the standard medium was modified with 1 ppm
(1.0 mg/l) benzylamino purine, 0.1 ppm (0.1 mg/l)
gibberellic acid, and lppm (1.0 mg/l) indole-3-butyric
acid. Plants were rooted directly from this proliferation
phase in a greenhouse under mist during the winter.

ST plants were set in the South Deerfield nursery in
the last two weeks of April, 1982. MP plants, which were
more tender, were set 2 weeks later to avoid cold damage
from late season frosts. The MP and ST plants were field
propagated in an identical manner using standard nursery
techniques including fumigation and pesticides to control
aphids. Plants were grown in a random arrangement on
adjacent blocks of land on a uniform Hadley fine sandy
loam soil. Dormant plants were dug in the winter and cold

stored at 30° F (-1° C).

14

Field testing. Field tests of these stocks were

 

conducted during 1983-1984 at the Horticultural Research
Center, Michigan State University, East Lansing. Dormant
runner plants from the MP and ST plants were planted in a
Conover loam soil. The MP plants were somewhat smaller in
size than the ST plants at planting. Conventional
cultural practices, including overhead irrigation (6), were
used Flowers were removed in the first year (1983). The
experiment consisted of a completely randomized design with
four plots per prOpagation method per cultivar. Each plot
was 12 ft (3.65 m) long with plants set initially at 1.5 ft
(46 cm) in the row, and 4 ft (1.22 m) on center. Plants
were trained to 1.5 ft (46 cm) wide matted rows.

Plant and crown numbers were recorded early in the 1984
season. At full bloom, total flower truss numbers per plot
were recorded. Plots were harvested every 3-4 days during
the period of June 18 to July 11, 1984. Plot yields and
total fruit numbers were obtained, and from these data,
average fruit weight and number of fruit per truss were

calculated.

Results and Discussion
The field performance of the MP and ST plants of six
cultivars is compared in Table 1. Overall, the MP plants
produced more runner plants than ST plants, and as a
result their yields were higher. There appeared to be

variation in the response of cultivars to micropropagation,

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co>Humc wum>fluaso wuuwnzmuum xflm mo mocmeuomuom pamflm

16
as 'Earliglow', 'Redchief', 'Bounty' and 'Scott' had
significant differences in plant number and/or yield,
while 'Guardian' and 'Honeoye' did not. In 'Earliglow' and
'Redchief' MP rows plant density was greater than eight
plants per foot of row. This amount of crowding may
have reduced fruit size in these rows as it has in other
studies with MP plants (9).

From a commercial grower standpoint, MP plants produced
45 percent more runners than ST mother plants. This
stimulation can be used to increase first-year yields
through rapid bed fill to optimum density. This can result
in an increase in yield; however, MP-originated runner
plants may cause overcrowding and fruit size can be
reduced. Thus, wider spacing for MP-derived stock may be
necessary for vigorously runnering cultivars.

No aberrant plants occurred among the first generation
daughter plants, and in the second year only one
'Earliglow' 'white-streak' chimeral mutant (9) was observed
in the planting. This plant was one out of approximately
4,000 descended from micropropagated plants.

Several studies have suggested that the stimulation of
runner production induced by micropropagation is temporary
and restricted to mother plants and their primary daughters
(7,9). Our results do not agree with their observations,
as randomly selected daughters from MP plants produced more

plants than those from standard beds. Cold may play a role

17
in this difference, as the plants evaluated in other
studies (7,9) were not subjected to a cold period as
were the plants in this study. Other workers have
indicated that cold storage may have a positive effect on
MP plant performance (1,3,4).

In conclusion, MP plants were vegetatively more
prolific two years after tissue culture. For some
cultivars, growers can use this altered growth habit to
achieve proper plant density for optimum yield in the first
year while reducing nursery costs by increasing planting
distance. We did not determine why some cultivars, e.g.,
'Honeoye', were less responsive to micropropagation than
others, but the increased variability in these rows could
have been genetic or compounded by environmental variation

during micropropagation or field handling.

Acknowledgements
Several people provided excellent technical assistance
during the course of this study: Peter Callow, Susan
Cheplick, Kathy Morimoto, Tom Petito, Marvin Pritts, Carol

Schumann and Jim Siefker.

Literature Cited

1. Aerts,J. 1979. "Evaringen met, langs microvermeerdering
bekomen, virusvrije aardbeiplanten." Meded. Fakult.
waetensch. Gent. 44:981-991.

2. Boxus, P. 1974. "The production cf strawberry plants by
in vitro micropropagation." J. Hort. Sci. 49:209-210.

18

3. Damiano, C. 1980. "Strawberry micropropagation." p.
11-22. In Proc. conf. on nursery production of fruit
plants through tissue culture-applications and feasibility.
USDA-SEA, Agr. Res. Results. ARR-NE-ll.

4. Damiano, C., W. Faedi and D. Cobianchi. 1979.
“Observazionin agronomiche su piante di fragola 'Gorella' e
'Aliso' ottenute dan colture" in vitro. p. 65-74. In:
S.O.I. Incontro nazionale sullan coltura della fragola.
Ferrara.

5. Hancock, J.F., M.P. Pritts and J.H. Siefker. 1984.
"Effect of runner removal and spacing on yield components
of strawberries." Crop Research 24:37-43.

6. Hull, J., J. Moulton and J. Flore. 1977. Growing
Strawberries in Michigan. Extension Bulletin E-682,
Cooperative Extension Service, Michigan State University.

7. Marcotrigiano, M., H.J. Swartz, S.E. Gray, D. Tokarcik,
and J. Popenoe. 1984. "The effect of benzylamino purine

on the in vitro multiplication rate and subsequent field
performance of tissue-culture propagated strawberry
plants." Adv. Strawberry Prod. 3:23-25.

8. Sansavini, S. and G. Gherardi. 1979. "Selezione
clonale e stabilita genetica di fragola micropropagate." p.
153-168. In: S.O.I. Incontro nazionale sulla coltura della
fragola. Ferrara.

9. Swartz, H.J., G.J. Galletta and R.H. Zimmerman. 1981.
"The field performance and phenotypic stability of
tissue-culture propagated strawberries." J. Amer. Soc.
Hort. Sci. 106:667-673.

SECTION II

ENHANCED VIGOR IN VEGETATIVE PROGENY OF
HICROPROPAGATED STRAWBERRY PLANTS

19

20
Abstract. Previous reports have indicated that the
physiological influence of in yitrg micropropagation is
limited to mother plants and their primary runner plants.
In this study, detached primary, secondary, and tertiary
runner plants of micropropagated plants all responded
differently than conventionally propagated plants of similar
age. Total flower number was significantly greater in all
runner plants of micropropagated 'Redchief' plants due to
increases in number of trusses and flowers per truss.
Anthesis was earlier in all runners of micropropagated
'Earliglow'. Fruit size was reduced in all runners of
micropropagated 'Redchief'. Micropropagated 'Earliglow'
also had significantly greater numbers of stolons. Reduced
numbers of flower trusses per plant and flowers per truss
were noted in tertiary runners of micropropagated
'Earliglow'.

Micropropagated mother plants of strawberry often
produce more flowers and runners than their
runner-propagated counterparts (4,5), but how long this
effect lasts is not clear.‘ Swartz et al (5) reported that
the increase in runnering ability of tissue culture (TC)
plants did not continue after the first flush of runner
production in the field, and Marcotrigiano, et a1. (4)
showed that within the planting year, micropropagation
increased runner production in mother plants and their

primary runner plants, but not in attached secondary and

21

tertiary runner plants. However, these studies examined
runner plants still attached to plants directly out of
culture, rather than detached, cold-hardened plants as they
are delivered to the grower. In a study where detached
runner plants of unknown age were compared, planting stock
from micropropagated plants of four of six cultivars had
significantly higher matted row densities (x = 63 percent)
than those derived from conventionally propagated stock (2).
The goal of this study was to compare the performance of
detached primary, secondary, and tertiary runner plants of
micropropagated (MP) and standard (ST) mother plants grown
under controlled conditions. MP and ST plants were produced
in 1984 according to previously described methods (2). In
May, MP and ST plants of the cultivars 'Earliglow' and
'Redchief' were planted in field plots 3.05 m long in a
completely randomized design. Plants were spaced 0.5 m in
the row and trained to matted rows 0.46 m wide and 1.22 m
between rows. The site was located at the Michigan State
University Horticultural Research Center on a well-drained
Conover loam soil with overhead irrigation. Blossoms were
removed after planting and standard cultural practices used
in Michigan were maintained.

Primary, secondary, and tertiary runner plants were
located in the plots in late November by tracing stolons
from the mother plants. Plants were then dug, cleaned,

trimmed of all leaves and stored -1° C for 14 weeks in

22
plastic bags. On March 12 of the following year, fresh
weight, crown diameter and root system length were recorded.
Plants from both propagation methods were of comparable size
and fresh weight with the exception of primary runner plants
of 'Redchief', which had significantly (p < 0.01; df = 17)
greater fresh weights than their micropropagated
counterparts (17.0 vs 11.5 g).

Ten to 15 plants from each cultivar and propagation
method were then potted in 20.3 cm diameter plastic pots in
a commercial peat and perlite potting mix and randomly
arranged on a greenhouse bench in the Plant Science
Greenhouses at Michigan State University. High pressure
sodium lamps 1.5 m above the pots provided supplemental
illumination of approximately 200 - 350 umol s'1 m;2
PPFD at plant level for 16 hours, and day/night temperatures
were maintained at approximately 24/18O C.

After an initial evaluation on March 19, numbers of
trusses, flowers, and runner plants were recorded every two
to three days from March 27 until April 29 and biweekly
thereafter until June 19. -During flowering, pollen was
applied among open flowers daily with a camel hair brush and
the first 50 ripe (90 percent red) fruit from each treatment
group were harvested.

MP and ST runner plants of the two cultivars differed in
sexual and asexual reproductive behaviors. In 'Redchief',

total numbers of flowers per plant were significantly

23
greater in all the runner plant generations of MP plants
when compared to ST plants (Table 1). Tertiary 'Redchief'
runner plants produced fewer flowers than primary ones, but
the percent difference among MP and ST plants remained
stable (50 - 62 percent). Fruit from all generations of MP
'Redchief' plants were significantly smaller than those from
ST plants. All progeny of MP 'Redchief' bloomed later than
ST plants, but the difference was significant only for
primary runner plants. No significant differences in
vegetative behavior were noted between ST and MP runner
plants of 'Redchief', although MP runner plants generally
had higher values.

Fifty percent bloom was occurred earlier in runner
plants of micropropagated 'Earliglow' in all generations,
but none of the other sexual traits showed consistent,
significant variations between ST and MP progeny. Primary
runner plants of MP plants had significanly more flowers per
truss and fewer trusses than ST plants, but this difference
Was not evident in tertiary runner plants. Runner plants
of micropropagated 'Earliglow' produced more stolons,
daughter plants per stolon and total daughter plants per
plant than did conventionally propagated runner plants
(Table 2), and this pattern was consistent across
generations.

Previous reports have suggested that the physiological

influence of micropropagation is limited to the

Table 1:

24

Sexual responses of detached primary (1°),-

secondary (2°) and tertiary (3°) daughter plants from
standard (ST) and micropropagated (MP) mother plants of strawberry.
Numbers in parentheses represent difference (%) between MP and ST plants.

 

 

Runner Plant Generation:
Propagation

Cultivar method 1° 2° 3°
Trusses per plant
Earliglow ST 1.6 1.2 1.3

MP 2.1 (31.3)* 1.7 (41.7)* 1.3 (0.0)
Redchief ST 3.0 2.4 1.8

MP 3.8 (26.7) 2.6 (8.3) 2.4 (33.3)
Flowers per truss
Earliglow ST 8.5 9.7 6.5

MP 6.9 (-18.8)* 7.6 (-21.6) 6.4 (—1.5)
Redchief ST 5.2 6.0 5.7

MP 6.4 (23.1)* 7.7 (28.3) 7.2 (26.3)
Flowers per plant
12mliglow ST 13.1 11.3 8.2

MP 14.2 (8.4) 12.8 (13.3) 7.7 (-6.1)
Redchief ST 15.4 13.1 9.6

‘ MP 23.2(50.6)*Y 19.8 (51.1)* 15.6 (62.5)*

Days to 50% bloomZ
Earliglow ST 9.2 8.5 10.5

MP 5.5 (-40.2)*** 5.6 (—34.1)* 5.4 (-48.6)
Redchief ST 10.0 10.9 11.4

MP 13.8 (38.0)** 11.7 (7.3) 13.0 (14.0)
Fruit weight
Earliglow ST 6.0 6.1 4.9

MP 6.2 (3.3) 5.7 (-6.6) 6.4 (32.0)
Redchief ST 6.0 5.5 6.0

MP 3.2 (—46.7)*** 4.1 (—18.2)* 4.1 (—31.7)

 

YPropagation methods significantly different at p

and p < 0.001 (***) by t-test.

zAfter planting

< 0.05 (*), p < 0.01 (**),

25

Table 2: vegetative response of detached primary (1°),
secondary (2°), and tertiary (3°) daughter plants from
standard (ST) and microprogated (MP) mother plants of strawberry.
Numbers in parentheses represent difference (%) between MP and ST plants

 

Runner Plant Generation:

 

Propagation
Cultivar method 1° 2° 3°

 

Stolons Per Plant

Earliglow ST 11.9 12.9 9.3

MP 16.7 (39.5)*2 19.2 (48.8)*** 20.6 (125.6)***
Redchief ST 10.4 13.1 12.6

MP 14.5 (39.4) 14.9 (13.7) 14.3 (13.5)
Plants per stolon
Earliglow ST 4.3 5.0 3.9

MP 5.9 (37.2)* 6.1 (22.6) 5.5 (41.0)*
Redchief ST 4.4 5.8 5.1

MP 6.1 (38.6) 6.6 (13.7) 6.8 (33.3)
Total runner plants
Earliglow ST 2.4 2.6 2.2

MP 3.0 (25.0) 3.2 (23.1)* 3.8 (72.7)***
Redchief ST 2.3 2.3 2.3

MP 2.5 (8.7) 2.3 (0.0) 2.2 (-4.3)

 

ZPropagation methods significantly different at P < 0.05 (*), P < 0.01 (**), or
P < 0.001 (***) by t-test.

26
micropropagated mother plant and primary runner plants
and, as a result, only the nurseryman and not the grower
would benefit from any increases in vigor due to the use of
MP foundation stock (4,5). Our results do not agree with
these conclusions as the runners of MP plants often behaved
differently than ST plants, and most of these differences
were consistent across generations. Only flowers per truss
in 'Earliglow' did not fit this pattern. Not all the
observed differences between MP and ST plants would benefit
the grower (e.g. smaller fruit size in 'Redchief'), but the
overall vigor of MP plants was generally higher.

Perhaps our results differ from those of previous
workers because we examined detached, cold-hardened plants
as they are delivered to the grower rather than runners
attached to mother plants directly from post-culture
acclimation (4,5). Damiano (3) and Aerts (1) have
demonstrated that cold storage prior to planting can improve
MP plant performance.

It is interesting that the two cultivars differed in
their responses to in yitré propagation. Sexual responses
were significantly increased in 'Redchief' by
micropropagation but not asexual processes, while in
general, the reverse was true for 'Earliglow'. Similar
differences in responses have been noted in other studies
(2,5). MP-enhanced vigor cannot last indefinitely as the ST

plants used in this study were descended from plants

27
originally cultured in vitro for disease elimination.
However, the data in Tables 1 and 2 imply that the MP effect
can extend beyond the primary runners in the first growing
season. The physiological basis of this enhanced vigor is

unknown.

Literature Cited

1. Aerts, J. 1979. Evaringen met, langs microvermeerdering
bokomen, virusvrije aardbeiplanten. Meded. Fakult.
waetensch. Gent. 44:981-991.

2. Cameron, J.S., and Hancock, J.F. 1985. The field
performance of strawberry nursery stock produced originally
from runners or micropropagation. Adv. Strawberry Prod.,
4:56-58.

3. Damiano, C. 1980. Strawberry micropropagation. In:
Proc. conf. on nursery production of fruit plants through
tissue culture-applications and feasiblity. USDA-SEA, Agr.
Res. Results. ARR-NE-ll.

4. Marcotrigiano, M., Swartz, H.J., Gray, S.E., Tokarcik,
D. and Popenoe, J. 1984. The effect of benzylamino purine
on the in vitro multiplication rate and subsequent field
performafice of tissue-culture propagated strawberry plants.
Adv. Strawberry Prod. 3:23 - 25.

5. Swartz, H. J., Galletta, G.J., and Zimmerman, R.H.
1981. The field performance and phenotypic stability,
of tissue-culture propagated strawberries. J. Amer.
Soc. Hort. Sci., 106:667-673.

SECTION III

THE INFLUENCE OF MICROPROPAGATION ON
YIELD COMPONENTS, DRY MATTER PARTITIONING
AND GAS EXCHANGE CHARACTERISTICS OF STRAWBERRY

28

29

Abstract. Asexual progeny of micropropagated (MP) plants
of the cultivars 'Earliglow' and 'Redchief' had higher plant
densities and yields than conventionally propagated (CP)
plants in matted rows. MP plants partitioned nine to ten
percent less of their total dry matter to leaf tissue. MP
'Redchief' increased its allocation to crowns and flower
trusses; 'Earliglow' to fruit. Leaf area per crown was
reduced in MP 'Earliglow' (49 percent) and 'Redchief'

(55 percent), and yield per crown was lower (32 percent) in
MP 'Redchief', but not in 'Earliglow'. Net photosynthetic
rate (mg C02 dm"2 hr"1 and ug C02 gm dr wt‘1 hr‘l) was
greater in MP 'Earliglow' but not in 'Redchief'. Stomatal
conductance was higher in MP 'Earliglow', but mesophyll
conductance was unchanged in both cultivars. Possible roles
of assimilate partitioning and demand and gas exchange I
behavior in MP plant performance are discussed.

Key words: Fraqaria x ananassa Duchesne,

 

micropropagation, phenotypic stability, field performance,

dry matter partitioning, net photosynthesis.

INTRODUCTION
Micropropagated (MP) strawberry plants are more vigorous
and productive than their conventionally-propagated (CP)

counterparts (Damiano, 1980; Swartz gt-al., 1981). Plants

30
set directly from ta ttttg culture form more stolons in
nursery beds (Marcotrigiano gt gl., 1984; Swartz gt gl.,
1981), and use of planting stock derived from these beds
can result in higher plant densities and increased yields
(Cameron and Hancock, 1985).

The yield component interactions of MP planting stock
and CP plants often vary, and differences in response have
been observed between genotypes (Cameron and Hancock, 1985;
Cameron gt gt., 1986). In a recent study, rows of MP
progeny of 'Redchief' and 'Earliglow' yielded more per unit
area than CP plants (Cameron and Hancock, 1985). However,
MP stock of 'Redchief' produced fewer fruit per crown and
lower yields per plant compared to CP stock, while no
significant differences were observed between MP and CP
'Earliglow' in these traits. These results suggest that
micropropagation may have genotype-specific affects on the
gas exchange rates and/or allocation patterns of
photoassimilates.

While the gas exchange characteristics of leaves from MP
strawberry plants during gt ttttg acclimization have
been previously described (Grout and Millam, 1985), there
have been no reports of the gas exchange properties and dry
matter partitioning patterns of these plants or their
progeny in the field. The purpose of this investigation
was to determine the influence of micropropagation on the

morphology, dry matter partitioning patterns and gas

31
exchange properties of field-grown 'Redchief' and
'Earliglow'; cultivars whose yield components are affected

differently by micropropagation.

MATERIALS AND METHODS

The experiment was conducted at the Horticultural
Research Center, Michigan State University, East Lansing.

MP and CP plants of the cultivars 'Earliglow' and 'Redchief'
were established in matted rows in a well-drained, Conover
loam soil. Flowers were removed in the planting year
(1984), and conventional cultural practices were maintained
(Hull gt gl., 1977). The experiment consisted of a
completely randomized design with four plots per propagation
method per cultivar. Each plot was 3.05 m in length with
plants initially set at 46 cm in the row and 1.22 m on
center. Plants were trained to matted rows 61 cm wide.

Dry matter partitioning patterns were determined in one
centrally located quadrat (0.29 m2) per plot. Ripe fruit
(90 percent red) were harvested every two to three days in
early June, and fresh and dry weights were obtained. After
harvest, plants in quadrats were dug, divided into leaves,
roots, crowns and flower trusses, and dried in an oven for
two weeks at 80° C.

Gas exchange, chlorOphyll, and weight per leaf unit area
measurements were made on single leaf samples from four
plants per quadrat. Gas exchange measurements were made

using an Analytical Development Corporation (ADC) C02

32
analyzer (Model LCA-2) equipped with a Parkinsdn broad leaf
chamber. Depletion of C02 by a 6.25 cm2 area of a single
leaf in the chamber was monitored at a flow rate of 0.4
liter min'l. Measurements were made on recently expanded
leaves (2nd or 3rd) in the upper canopy. Readings were
taken in full sunlight on a clear, sunny day at 50 percent
harvest between 1000 and 1200 HR at 25 * 1° C. Gas exchange
parameters were calculated using standard formulae (Moon and
Flore, 1986).

A 0.625 cm2 leaf disk was immersed in 5 ml n,n
dimethylformamide. Chlorophylls a and b and
protochlorophyll content were determined using a
spectrophotometric assay and formulae described by Moran
(1982). Weight per leaf unit area was calculated based on

the mean of two, 0.625 cm2 leaf disks per leaf.

RESULTS

Plant and crown densities were significantly higher in
MP quadrats (Table 1), but crown diameter was not influenced
by micropropagation. Mean.leaf number and individual and
total leaf areas were significantly lower in MP plants.
Leaf area index wassignificantly greater in MP 'Redchief',
but not in MP 'Earliglow'. Neither flower trusses per crown
nor fruit size were affected by micropropagation in either
cultivar. Yield per quadrat was significantly greater in MP

plants of both cultivars, whereas yield per plant and per

33

Table 1: Yield components of plants in matted
row quadrats of micropropagated (MP) and conventionally
propagated (CP) 'Earliglow' and 'Redchief' strawberry.

 

 

 

 

 

Earliglow Redchief
Yield
component MP CP MP CP
vegetative
Plants/.093 m2 16.4**2 8.3 17.1*** 5.6
Crowns/.093 m2 21.8* 11.3 27.4*** 9.4
Mean crown
diameter (cm) 1.2 1.2 1.1 1.1
Individual leaf area
(cm2) 61.9* 76.1 51.2* 65.1
Leaves/crown 4.2* 6.7 3.5* 6.5
Leaf area/crown
(m2) 0.026* 0.051 0.018* 0.04
leaf area index
(m2 leaf area/
.093 m2 row) 6.1 6.2 5.3* 4.3
Reproductive
Flower trusses/crown 1.2 , 0.9 1.1 1.2
Individual fruit
weight (g) 4.9 _ 4.7 4.7 4.5
Fruit/crown 4.5 4.3 3.5* 5.5
Yield/crown (g) 22.3 20.1 16.6* 24.5
Yield/plant (g) 30.1 26.4 26.7* ‘ 44.5
Yield/quadrat (kg) 0.88* 0.39 0.80* 0.42
z*,**, *** =Propagation means within cultivars significantly different by

t-test, p < .05,.01,.001 respectively.

34
crown and fruit number per crown were lower in MP 'Redchief' but not in MP
'Earliglow'.

Tbtal dry matter accumulation was significantly lower (49 percent) in plants
of MP 'Redchief' but not in 'Earliglow'; however, higher reproductive effort
(percentage of dry matter in reproductive structures) was expended by MP
'Earliglow' (Table 2). Leaf dry matter accumulation was strongly influenced
by micropropagation as MP plants partitioned nine to ten percent less of their
total biomass into leaves than did CP plants. MP plants of 'Redchief'
partitioned more of their dry matter into crowns and trusses, while MP
'Earliglow' had significantly higher allocation to fruit.

weight per leaf unit area was significantly lower (11 - 12 percent) in MP
plants than in GP plants (Table 3). No differences were noted in content of
Chlorophylls a, b, protochlorophyll or total chlorophyll. Higher rates of
net photosynthesis were noted in only MP 'Earliglow' on both a leaf area and
per leaf area dry weight basis, however, total plant assimilation was
significantly lower in MP plants of both cultivars. No significant
differences were found in mesophyll conductance (gm) between MP and CP leaves
of either cultivar, although stomatal conductance (gs) was higher in MP

'Earliglow'.

35

Table 2: Biomass allocation in plant
structures of micropropagated (MP) and conventionally
propagated (CP) 'Earliglow' and 'Redchief' strawberry

 

 

 

 

 

Earliglow 'Redchief
MP cp MP "“ cp
Leaves 37.1** 46.4 34.1** 43.9
Roots 11.4 11.1 10.3 9.2
Crowns 13.6 13.2 14.4** 11.9
Truss 5.4 4.0 7.7*** 4.1
Fruit 32.4* 25.4 33.7 31.1
Reproductive
effort 37.8* 29.4 41.3 35.1
Tetal dry
matter per
plant (g) 10.4 13.2 9.1* 17.7
m

=Propagation means significantly different by t-test, p < 0. 05,
0. 01, and 0. 001 respectively.

Table 3: Gas exchange, weight per leaf unit area, and total
chlorophyll in matted row quadrats of micrOpropagated (MP) and
conventionally propagated (CP) 'Earliglow' and 'Redchief' strawberry.

 

 

Earliglow Redchief

Parametery MP CP MP CP
weight per leaf unit area

(mg cm'2) 6.7**2 7.5 6.7** 7.6
Total chlorophyll (ug cm-Z) 5.1 5.2 4.3 3.6
Pnet - **

(ug 002 gm at wt-lhr-l) 3.2 2.2 3.0 2.9
Pnet *

(mg 002 dm-Zhr-l) 21.3 16.6 19.7 21.4
Pnet

(mg c02 plant 1hr‘1) 74.2* 116.6 58.0**l 144.5
95 . . *

(mulllmols s-l m2) 275. 231. 261. 256.
9m

(millimols s-l m2) 122. 93. 111. 127.

 

yPnet = net photosynthesis, gS = stomatal conductance, gm = mesophyll
conductance.

2*,** *** =Mean5‘within a cultivar significantly different by t-test
p < 0. 05, 0. 01, 0. 001, respectively.

36

DISCUSSION

As observed previously (Cameron gt gl., 1985), MP
derived planting stock of both 'Earliglow' auul 'Redchief'
produced higher yields per unit area than CP stock. This
increase was associated with higher plant densities in both
cultivars. Yields per plant were significantly lower in MP
than in CP 'Redchief', but not in 'Earliglow'. MP
'Redchief' also showed a dramatic (36 percent) reduction in
the number of fruit per crown; fruit numbers were unchanged
in MP 'Earliglow'. These differences arose even though the
plant densities of MP 'Earliglow' and 'Redchief' were not
significantly different.

Differences in tolerance to density have been described
in other cultivars (Swartz gt gl., 1982; Hancock gt gl.,
1984). Shading may have played a role as 'Redchief' had a
higher leaf area index (LAI) in MP than in CP rows, while
the LAI of 'Earliglow' was unchanged. Reduced light levels
may have decreased flower bud initiation and fruit set in
'Redchief'(Anderson and Guttridge, 1976; Guttridge and
Anderson, 1973). It is also possible that the gas exchange
properties of 'Earliglow' are less sensitive to shading than
are those of 'Redchief'.

MP plants of 'Earliglow' and 'Redchief' allocated a
higher proportion of their total fixed carbon to
reproductive structures than CP plants. MP 'Earlighmw

allocated significantly more dry matter to fruit, while MP

37
'Redchief' allocated a significantly higher proportion of
its energy to crowns and trusses. Strawberry fruit are the
major sink for dry matter in the plant (Forney and Breen,
1985; Olsen gt a_l., 1985). Forney and Breen (1985) have
demonstrated that dry matter accumulation in strawberry
fruit can create a greater demand for assimilates than the
plant can maintain; this forces the plant to draw from
reserve carbohydrates during fruitingu (Consequently,
assimilate demand by strawberry fruit can influence dry
matter allocation to developing leaves (Forney and Breen,
1985; Schaffer gt gt., 1985). In this study, significant
reductions (nine to ten percent) in dry matter allocation
to leaf tissue in MP plants was manifested in reductions in
leaf numbers, leaf area, and weight per leaf unit area.
Total leaf area per crown was reduced 49 percent in MP
'Earliglow', and 55 percent in MP 'Redchief'. The presence
of fruit can enhance rates of photosynthesis in strawberry
(Choma e_t a__l., 1982; Walkowa gt a_l_., 1974), and may be a
result of increased assimilate demand (Forney and Breen,
1985). MP 'Earliglow' had less leaf surface area than CP
'Earliglow'and yields per crown were unchanged; this may
have created a greater assimilate demand and increased
Pnet in MP plants. Yield per crown was significantly
(p < 0.05) reduced (32 percent) in MP 'Redchief', and
reduced sink strength in these plants may not have been

sufficient to increase assimilate demand and Pnet-

38

Pnet could have been influenced by a number of other
factors in this study. Stomatal function, distribution and
number are important limiting factors in plant gas exchange
and are altered by the 12 ytttg environment (Donnelly and
Vidaver, 1984); however, strawberry leaves developing after
acclimation gradually revert tonormal anatomy and function
(Fabbri gt gl., 1986; Grout and Millam, 1985). In a recent
report (DeJong,1986), describing the effects of the presence
of peach fruit on rates of Pnetr it was concluded that
this influence was primarily related to stomatal behavior;
Pnet and 95 increased but gm was unchanged. SimilarLy,
while no changes in gm were observed in either cultivar in
this study, increases in both Pnet and gS were observed in
MP 'Earliglow' but not in 'Redchief'. The nature of
stomatal limitations induced by fruit are poorly understood
(DeJong,1986).

There are also many biochemical influences which have
direct and indirect effects on Pnet’ levels or activity of
ribulose-l, 5-bisphosphate carboxylase/oxygenase and
regeneration of 1135 substrate, photorespiration, and
triose phosphate utilization are examples of potentially
rate-limiting factors (Sharkey, 1985). While no significant
differences were observed in mean rates of gm in this study,
biochemical or diffusional differences could occur in leaves
of MP plants under nonfruiting conditions; gas exchange

behavior of deblossomed strawberry plants is different from

 

39
that of fruiting plants (Choma gt gl,, 1982; Forney and
Breen, 1985).

The physiological basis of the phenotypic variability
cmmerved in nucropropagated strawberry cultivars has not
been determined. While enhanced vigor in micropropagated
strawberry plants can last beyond the first year and the
micropropagated mother plant (Cameron and Hancock, 1986;
Cameron gt gt” 1985), it is not known whether there are
lasting physiological changes effected in the plant; no one
has demonstrated physiological or morphological changes IJI
these plants 12 yttg which are directly attributable to some
aspect of the 12 ytttg environment. The 25 gtttg influence
of i_n vitro applied plant growth regulators could be
considered a possible cause, as relative media levels of
auxins, cytokinins, and gibberellins can have a pronounced
effect on the growth and development of strawberry 12 ttttg
(Waithaka gt a_l_., 1978) and _i_n_ 1129. (Dennis and Bennett,
1969; Tafazoli and Shaybany, 1978). However, in one study
(Marcotrigiano gt a_l., 1984), varying i_n \Li_t_rg levels of
cytokinin did not alter stolon production in the field.
Other factors such as enhanced nutritional status after
culture could have played a role in the difference between

MP and CP plants, but no information exists on the elemental

composition of micropropagated strawberry plants.

40
REFERENCES

Anderson, H. M. and Guttridge, C.G., 1976. Fruit yield
component of strawberries grown in solid beds. (1. Hortic.
Sci., 51:215-223.

Cameron, J.S. and Hancock, J.F., 1986. Enhanced vigor in
vegetative progeny of micropropagated strawberry plants.
HortScience, 21(5). In press.

Cameron, J.S., Hancock, J.F., and Nourse, T.M., 1985. The
field performance of strawberry nursery stock produced
originally from runners or micropropagation. Rec. Adv.
Strawberry Prod., 4:56-58.

Choma, 1LJ3., Garner, J.L., Marini, R.P. and Barden, J.A.,
1982. Effects of fruiting on net photosynthesis and dark
respiration of 'Hecker' strawberries. HortScience,
17:212-213.

Damiano, C., 1980. Strawberry micropropagation. p.11-22,
In Proc. conf. on nursery production of fruit plants
through tissue culture - applications and feasibility.
USDA-SEA, Agr. Res. Results, ARR-NE-ll. -

DeJong, lLJ4., 1986. Fruiting effects on photosynthesis in
Prunus persica. Physiol. Plant., 66:149-153.

 

Dennis ;h:., F.G., and Bennett, H.O., 1969. Effects of
gibberellic acid and deflowering upon runner and
inflorescence development in an everbearing strawberry.
J. Amer. Soc. Hort. Sci., 94:534-537.

Donnelly, D.J. and Vidaver, W.E., 1984. Leaf anatomy of red
raspberry transferred from culture to soil. J. Amer.
Soc. Hort. Sci., 109:172-176.

Fabbri, A., Sutter, E., and Dunston, S.K., 1986. Anatomical
changes in persistent leaves of tissue-cultured strawberry
plants after removal from culture. Scientia Hortic.,
28:331-337.

Forney, C.E., and Breen, P.J., 1985. Dry matter partitioning
and assimilation in fruiting and deblossomed strawberry. J.
Amer. Soc. Hortic. Sci., 110(2):181-185.

Grout, B.W., and Millam, S., 1985. Photosynthetic
development ofmicropropagated strawberry plantlets following
transplanting. Ann. Bot., 55:129-131.

Guttridge, C.G., and Anderson, H.M., 1973. The relationship

41

between plant size and fruitfulness in strawberry in
Scotland. Hort. Res., 13:125-135.

Hancock, J.F, Moon, J.W., and Flore, J.A., 1984. Within-row
spacing auul dry weight distribution in two strawberry
cultivars. HortScience, 19:412-413.

Hull, J., Moulton, J. and Flore, J., 1977. Growing
strawberries in Michigan. Extension Bulletin E-682,
Cooperative Extension Service, Michigan State University.

Marcotrigiano, M., Swartz, H.J., Gray, S.E., Tokarcik, D.,
and Popenoe, J., 1984. The effect of benzylamino purine on
the lJl'VitrO multiplication rate and subsequent field
perfofmance of tissue-culture propagated strawberry plants.
Adv. Strawberry Prod., 3:23-25.

Moon Jr., J.W., and Flore, J.A., 1986. A BASIC computer
program for calculation of photosynthesis, stomatal
conductance, and related parameters in an open gas exchange
system. Photosynth. Res. In press.

Moran, 1%., 1982. Formulae for determination of
clilclrc3p11y'll.OLls pzigtnelnt s elctlracztead v:it:h
N,N-dimethylformamide. Plant Physiol., 69:1376-1381.

Olsen, J., Martin, L.W., Pelofske, P.J., Breen, l?uJ., and
Forney,C.F., 1985. Functional growth analysis of the
strawberry. J. Amer. Soc. Hort. Sci., 110:89-93.

Schaffer, B., Barden, J.A., and.VfiJliams, J.M.,1985.
Partitioning of [14CJ-photosynthate in fruiting and
deblossomed day-neutral strawberry plants. HortScience,
20:911-913.

Sharkey, T.D., 1985. Photosynthesis in intact leaves of
C3 plants: physics, physiology and rate limitations.
Bot. Review, 51:53-105.

Swartz, H.J., Galletta, G.J. and Zimmerman, R.H., 1981.
Field performance and phenotypic stability of tissue
culture-propagated strawberries. J. Amer. Soc. Hort. Sci.,
106:667-673.

Swartz, IIHJ., Walsh, C.S., Geyer, A.F., Douglass, L.,
Galletta, G.J. and Zimmerman, R.H., 1982. Plant crown
competition in strawberry matted rows. Rec. Adv. Strawberry
Prod., 1:6-11.

Tafazoli, 13. and Shaybany, B., 1978. Influence of
nitrogen,deblossoming, and growth regulator treatments on
growth, flowering, and runner production of the 'Gem'

"‘"nq

 

42

everbearing strawberry. J. Amer. Ekxz. Hort. Sci.,
103:372-374.

Waithaka, l(., Struckmeyer, B.E. and Dana, M.N., 1978.
Hormonal control of strawberry axillary bud development £2
vitro. J. Amer. Soc. Hort. Sci., 105:428-430.

Wolkowa, E., Antoszewski, R. and Poskuta, J., 1974. CO
exchange rates of strawberry plants during fruiting. Frui
Sci. Rpt., 1:18- 25.

SECTION IV

THE INFLUENCE OF LEAF
DENSITY AND SHADING ON GAS EXCHANGE
CHARACTERISTICS OF NICROPROPAGATED STRAWBERRY PLANTS

43

44

AbMuacL. Gas exchange behavior in response to
increasing leaf density (leaf number per 0.093 m2) was
compared in matted rows of micropropagated (MP) and
conventionally propagated (CP) 'Earliglow' and 'Redchief'.
Highly significant (p < 0.001) negative, linear correlations
were observed between weight per leaf unit area and leaf
density in MP cultivars. Significant, negative linear
correlations were also observed between net photosynthetic
rate (Pnet)' stomatal conductance (gs), mesophyll
conductance (gm) and leaf density in CP, but not MP
cultivars. The effects of shading on potted 'Earliglow'
and 'Redchief' MP plants and their first and second-year
field grown runners were compared. While no differential
effects of shading were observed, Pnetr gs, gm,
transpiration (E) and chlorophyll content declined with
successive seasons of field propagation. Leaf number and
surface area, petiole length, and weight per leaf unit area
were less in MP plants compared to their asexual progeny.
Quantum yield, carboxylation efficiency, and 9m response
to increasing ambient C02 levels were greater in MP versus
CP 'Earliglow'; these differences were not observed in
'Redchief'. Higher maximum rates of Pnet were obserwai
in deblossomed MP cultivars compared to their

field-propagated progeny.

45

Micropropagated (MP) strawberry cultivars commonly have
higher yields per acre in matted rows than their
conventionally propagated (CP) counterparts due to higher
plant densities (4,5,21). However, these higher densities
can reduce individual plant yields (4,5). One limitation to
plant productivity imposed by high plant density could be a
reduction le‘the penetration of photosynthetically active
radiation through the leaf canopy (6,1,10). The influence
of shading (n1 leaf architecture, dry matter accumulation,
chlorophyll content and gas exchange have been studied 1J1

apple (2), Peach (14), sour cherry (19), Fragaria virginiana

 

Duchesne, tine meadow strawberry (13), and Fragaria vesca,

 

the woodland strawberry (6,7). Shaded plants have thinner
leaves, lower weight per leaf unit area, more chlorophyll,
and lower rates of photosynthesis.

In a previous study (4), individual plant yields
remained constant in one MP cultivar ('Earliglow') at
high density, while they were reduced in another
('Redchief') compared to GP plants. Progeny of
micropropagated plants of both 'Earliglow' and 'Redchief'
allocated 9 - 10 percent less of their dry matter to leaves.

These plants had only half the leaf area of CP plants due to

46
reduced leaf numbers and areas. Alterations in yield
component relationships (4,21), photoassimilate allocation
patterns, or gas exchange properties (4) could have played a
role 111 the greater tolerance of MP 'Earliglow' to higher
plant densities. The purpose of this study was to determine
whether leaf density and shading have a differential effect
on the gas exchange properties of MP and CP 'Earliglow' and

'Redchief'.

Materials and Methods

Two experiments were undertaken at the Hortnnfltural
Research Center, Michigan State University, East Lansing,
between 1984-1986. Experiment 1 was a field study examining
the influence of leaf density on gas exchange, while
experiment 2 examined the affects of shading on potted,
deblossomed plants.

Plants of the cultivars 'Earliglow'(E) and 'Redchief'(R)
were micropropagated (MP) and conventionally propagated (CP)
as previously described (5). Throughout this discussion,
subscripts of 0, 1, and 2 will be used to denote plants
directly from i_n 2% culture (E0, R0), first generation
asexually-propagated field progeny of micropropagated plants
(E1, R1), and second generation field progeny (E2, R2). CP
plants in this discussion will refer to those which have not
been propagated from MP plants within the previous three

seasons (E2, R2).

 

47

Gas exchange measurements were made using an Analytical
Development Corporation (ADC) C02 analyzer (Model LCA-2)
equipped with a Parkinson broadleaf chamber. Depletion of
c02 by a 6.25 cm2 area of a middle leaflet in the chamber
was monitored at a flow rate of 0.4 liter min'l. In
Experiment 1, photosynthetic measurements were made on 2
fully expanded leaves per plant; 1 in the upper canopy and 1
in the lower canopy. In Experiment 2, one fully expanded
leaf (2nd or 3rd) was measured from each plant. In
Experiment 2, C02 response was measured using an open gas
analysis system described previously (9,19). All gas
exchange parameters were calculated using standard formulae
(16).

Chlorophyll was extracted from a 0.625 cm2 leaf disk
in 5 ml n,n dimethyl formamide. Levels of Chlorophylls a, b
and protochlorophyll were determined using the
spectrophotometric assay and formulae described by Moran
(17). Weight per leaf unit area was calculated based on the
mean of two, 0.625 cm2 leaf disks per leaf.

Experiment 1. Greenhouse-acclimated E0, R0 and dormant

 

CP plants of both cultivars were planted 1J1 Late Spring,
1984, in a well drained Conover loam soil. Flowers were
removed in the planting year and conventional cultural
practices were maintained (12). The experiment consisted of
a completely randomized design with three plots per

propagation method per cultivar. Each plot was 3.05 m in

48
length with plants initially set at 46 cm1:U1 the row and
1.22 m on center. Plants were trained to matted rows 60 cm
wide.

When 50 percent of the fruit had been harvested in
the first bearing year, seven subplots (0.093 mg) within
each plot were selected which appeared to represent a range
of leaf densities. Gas exchange measurements were made on
two leaves from one plant in the center of the subplot.
Leaf number per subplot was recorded and correlated with dry
weight per leaf unit area (WLA), chlorophyll content, INN:
photosynthesis (PN), and stomatal (gs) and mesophyll
(9m) conductances. Readings were taken under full sun on two
consecutive, clear sunny days between 1000 and 1200 EUR at
25110 C.

Experiment 2. Plants of both cultivars and all

 

propagation generations (E0, E1, E2 and R0, R1, R2) were
potted in 20.3 cm plastic pots in a commercial peat and
perlite potting mix in late spring, 1986. E0 and R0 plants
were greenhouse acclimated and all field progeny were
dormant. Plants were arranged randomly on a greenhouse
bench 15 cm apart. All blossoms were removed and the plants
were allowed to develop three to four leaves under
greenhouse conditions for hone month prior to implementing
shading treatments. These leaves were tagged and not used

in the experiment.

49

Shading treatments were created by covering a pipe frame
structure with black polypropylene shade fabric (A.H.
Hummert Co., St. Louis, MO) which transmitted an average of
36 percent, 21 percent, or 9 percent photosynthetically
active radiation (PAR) as measured by the ADC LCA-2. A
previous study using this shading structure established that
ventilation prevented significant differences in temperature
and relative humidity between treatments, and that the
shading material reduced all wavelengths in the 380 - 750 nm
range equally (14). A fourth group of plants was grown
unshaded adjacent to the shading structure to achieve full
solar radiation. Plants of both cultivars from all
propagatitni treatments were divided equally among the
shading treatments and arranged in a random manner.

Average gas exchange values were determined at a
saturating level of PAR (1200 umol s’l m'z) under a high
pressure sodium lamp at a temperature of 26*1° C. Gas
exchange measurements were made on single leaves from five
plants per cultivar per treatment group. Each leaf was then
sampled for chlorophyll and WLA. Leaf areas, petiole
length, and leaf numbers per plant were obtained from three
plants per cultivar per treatment. The areas of three
randomly selected leaves from each plant were measured
using a LI-COR Model LI300 leaf area meter (LI-COR INC.,

Lincoln, Nebraska). Data from each cultivar were analyzed

50
as a two-way analysis of variance in a factorial experiment
with mean separation by Duncan's Multiple Range Test.

Light response of plants in all treatments was measured
using three plants per cultivar per treatment. One leaf per
plant was subjected to a range of light levels by reducing
full sunlight with neutral density filters. Six to 13 light
levels were used per replication. Readings were taken
adjacent to time shading structures under full sun at
28*2° C. Quantum yield was calculated for plants grown
under full sun by correlating PAR levels below 250 umol
s"1 m"2 with PN, as light response in this region was
linear. The effect of ambient C02 concentration on two
propagation generations of both cultivars (E0, R0 and E2,
R2) grown under full sun was tested in the laboratory.
Single leaflets were placed in plexiglass chambers at 25110
C and 1000 umol s‘"1 m"2 PPF. Plants were first equilibrated
at 330 umol mol'1 C02 and 21 percent 02 and then exposed
to various levels of C02 by adding C02 from a standard tank
(5 percent) to outdoor air which had been scrubbed of
C02 with soda lime (19). Carbon dioxide concentrathmus
ranged from 90-420 umol mol'1 and were nmmdtored on the
reference side of a Beckman 865 infrared gas analyzer with
the ADC LCA-2 C02 analyzer.

Results

Experiment 1. Highly significant (p < 0.001) negative

 

linear correlations were observed between WLA and leaf

51
number per subplot in MP plants of both cultivars (Table 1).
Significant, negative linear correlations were also observed
between leaf density and Pnet' gs, and 9m in CP cultivars,
but not in MP plants. On a dry weight per leaf area basis,
net photosynthesis was positively correlated with leaf
density in MP 'Earliglow', but not in MP 'Redchief' (Figure
1). No significant (p < 0.05) relationships between total
chlorophyll or its components and leaf number gnu: subplot
were observed (data not shown).

Weight per leaf unit area decreased with increasing leaf
canopy density at a greater rate in 'Earliglow' than in
'Redchief' (slopes significantly different, p < 0.01).
Stomatal and mesophyll conductances were both positively
correlated (p < 0.001) with Pnet in an identical manner in
both cultivars and propagation methods (data not shown).

Experiment 2. There were no significant interactions

 

between propagation generations and shading treatments for
gas exchange and chlorophyll data, thus shading treatment
data were pooled and propagation generation means were
compared. Gas exchange values were significantly greater
for plants directly from it gtttg culture than for field
grown progeny of MP plants (Table 2). Maximum rates of
photosynthesis declined with successive seasons of field
propagation in both cultivars.

Differences in chlorophyll content or its components

were less on a per leaf surface area basis than on a per dry

52

Table 1: Correlation coefficients of leaf density
(leaves per 0.093 m2) versus weight per leaf unit area
and gas exchange measurements in matted rows of MP and

CP 'Earliglow' and 'Redchief'strawberry.

 

 

 

Earliglow Redchief
MP ST MP sT

weight per leaf unit

area (mg cm-Z) -0.69***z -0.30 -0.46*** -0.23
Pnet

(mg C02 dm-Zhr'l) -0.06 -0.47:: -0.14 -0.37:
93 (cms'l) -0.01 -0.43 —0.18 -0.35
gm (cms-l) -0.17 -0.50*** -0.17 -0.39*
z *,**,*** = p < 0.05, 0.01, 0.001, respectively, n = 42

53

Table 2: Gas exchange characteristics of 'Earliglow'
and 'Redchief' strawberry plants of three propagation
generations (PAR = 1200 umol s'1 m-2)w

 

 

 

 

 

'Earliglow' 'Redchief'
Gas exchange
Parameterx Boy E1 E2 R0 R1 R2__
Pnet
(mg 002 dm-Zhr’l) 21.1az 16.9b 15.0c 21.9a 17.96 15.9c
Pnet

(ug coz gm dr wt'lhr‘l) 5.1a 3.5b 3.0c 4.8a 3.1b 2.9b

g

S

(millimols s-l m7?) 203.2a 177.0b 171.7b 198.4a 186.46 183.5b
E

(millimols s-l m7?) 6.7a 6.2b 6.1b 6.6a 6.26 6.06
gm. .

(milllmols s'l m'2) 50.7a 40.56 34.7c 53.5a 42.16 36.7c

 

wMeans of 1 single leaf measurement from 5 plants per treatment.

XPnet, net photosynthesis; gs, stomatal conductance; E, transpiration;
gm, mesophyll conductance.

YSee explantion of abbreviations in text.

zMeans within a row and cultivar not followed by the same letter are
significantly different by DMR, p < 0.05.

54

Figure 1: Affect of leaf density (number of leaves per
0.093 m2) on net photosynthesis (PN) on a weight per leaf
unit area basis in matted rows of micropropagated (————) and
conventionally propagated (----) 'Earliglow' (A) and
'Redchief' (B). Each point represents a measurement of one

leaf with 21 replications per propagation treatment.

PN (pg COa mg dr wt"hr")

PN (pg 00, mg dr wt“hr")

55

 

 

 

 

 

 

 

 

 

 

 

 

4.0 .
0 MP

3.5- " ‘3" ’EOTOWO‘HO'OZG
3.0- . ' .
2.5-
2.0-
1.5-
1.0-

.. I.
0-5 ' {2239430935300w'0‘7
°'°s 1'01'5 26 26'??? 4'0 4'5 .6
4.0 B , ,
3.5- 3 c“: ”#:ESWQHO'W
3.0- , . ° - ' .
2.5..
2.0-
1.5-
1.0-
0.5-
”. 1'6 (‘5 2'6 25‘}??? 4'0 26 86

 

Leaf Density (leaves per 0.093 m')

56

weight basis (Table 3). MP plants contained higher levels
of chlorophyll and its components compared to their field
grown progeny. Higher chlorophyll content could indicate a
greater light harvesting capacity in MP plants. Quantum
yield was significantly greater in E0 compared to E2 plants
grown under full sun (slopes significantly different, p <
0.05), but no differences in quantum yield were observed
between MP and CP 'Redchief' (data not shown).

The effects of shading on leaf characteristics of MP and
CP plants of both cultivars are exemplified by the data
presented in Table 4. MP plants had fewer leaves with less
surface area, shorter petioles, and less weight per leaf
‘unit area than their asexual progeny under shaded and
nonshaded conditions (Table 4). A similar pattern was
observed in a previous study (4).

Shaded plants of both cultivars had lower maximum rates

of P (Table 5), and their response to increasing PAR was

net
also similar (See Figures 2a, 3a for typical responses).

Maximum rates of P g& and 9m were higher in MP plants

net'
than in second generation plants in both cultivars (Figure
2 and 3). Across all treatments, light compensation points
were below a PAR level of 100 umol s'1 m'2 and did not
appear to vary appreciably.

Response of MP and second generation plants grown under

full sun to increasing levels of ambient C02 are shown in

Figures 4 - 6. On a per leaf surface area basis,

57

Table 3: Chlorophyll content of leaves from 'Earliglow'

and 'Redchief' strawberry plants of three propagation generations

 

 

 

 

 

'Earliglow' 'Redchief'
Pigmenty Eoy E1 E2 R0 R1 R2
Chlorophyll a
(mg dm'z) 3.1az 3.1a 2.8a 3.6a 3.3a 2.96
Chlorophyll a
(ug mg dr wt-l) 7.5a 6.56 5.7c 7.8a 6.0b 5.46
Chlorophyll b
(mg dm'Z) 1.1a 1.1a 1.1a 1.2a 1.2a 1.06
Chlorophyll b
(ug mg dr wt-l) 2.5a 2.3a 2.3a 2.5a 2.16 1.96
Chlorophyll a/b
ratio 2.9a 2.8a 2.5b 3.1a 2.9b 2.8b
Total chlorophyll
(mg dm‘z) 4.2a 4.2a 3.9a 4.7a 4.5a 3.96
Total chlorophyll
(ug mg dr wt-l) 10.0a 8.86 8.06 10.3a 8.16 7.0c

 

YSee explanation of abbreviations in text.

2Mean of 1, 0.625 cm2 leaf disk from 5 plants per treatment averaged over
4 shading treatment levels; means within a row and cultivar not followed
by the same letter are significantly different by DMRT, p < 0.05.

58

Table 4: Characteristics of leaves from
'Earliglow' and 'Redchief' strawberry plants
of three propagation generations grown under 100% and 21% full sun.

 

 

 

 

 

'Earliglow' ' 'Redchief'

BOW E1 E32 80 R1 R2.
100% sun
Leaf number
per plantx 4.7az 6.0b 7.0c 5.7a 6.36 7.0c
Individual
leaf area (cm2)x 68.9a 89.26 88.16 82.2a 105.06 129.1c
Weight per leaf
unit area (mg cm-2)Y 6.0a 7.16 6.96 6.6a 8.26 7.66
Petiole length
(cm)X 7.8a 10.66 9.46 9.2a , 13.36 14.26
21% s 1:
Leaf number
per plant 5.0a 6.0b 5.6b 5.3a 6.3c 5.8b
Individual
leaf area (cm2) 100.4a 149.06 150.16 75.2a 119.66 119.26
weight per leaf .
unit area (mg cm-Z) 3.6a 4.1a 4.46 3.9a 5.36 4.5ab
Petiole length
(cm) 11.5a 15.66 15.6b 12.7a 16.46 17.36

 

wSee explanation of abbreviations in text.

XMean of 3 samples from 3 plants per treatment per cultiVar.

YMean of 2, 0.625 cm'2 leaf disks per leaf, one leaf per plant; 5
replications per treatment per cultivar.

zMeanswithin a row and cultivar not followed by the same letter are
significantly different by DMRT, p < 0.05.

59

Table 5: Maximum rates of net photosynthesis (PN) in 'Earliglow'
and 'Redchief' strawberry plants grown under different light levels.
Measurements made at PAR = 1200 umol 5'1 m’2 and 26:10 C

 

Net photosynthesis (mg C02 dm’zhr‘l)

 

 

Sunlight 'Earliglow' 'Redchief'
(%)
100Y 19.8az 20.3a
36 17.7b 19.5a
21 16.0c 17.8b
9 V 17.0bc 16.8bc

 

yMean of 1 single leaf measurement from 15 plants per shading treatment;
averaged over 3 propagation generations.

zMeans within a column and cultivar not followed by the same letter are
significantly different by DMRT, p < 0.05.

60

Figure 2: Response of gas exchange parameters of
micropropagated (MP (A, C, E) and conventionally propagated
(CP) (B, D, F) 'Earliglow' plants grown under full sun tx>
different levels of photosynthetically active radiation
(PAR). Gas exchange parameters: net photosynthesis (PN)
(A,B), mesophyll conductance (gm) (C, D) and stomatal
conductance (gs) (E, F). Different symbols represent

replications; 6 - 10 measurements per replication.

 

 

 

 

 

 

 

 

 

 

 

 

a , a
A . o. B
244 o . 24-
is . .- ..
5" Ili o I“ ' .
8 82" ° . ‘ '2‘ o .
a 0 o
E o
v .‘ . .J o
E °‘ . .' .
4" 0.. ‘d : o
:0 360 do no? 1500 (5'00 1800 2100 30* :50 050 050 (2'00 1500 1700 2100
70 10
eo-c :. - eo-D
8‘50- ° . 5°“
E . .
I. oo- ' 40-
ca 0‘ O O.
E a . . ‘
5 3° . 3° , .
new 3 20d 0. .. .
go. .0 10‘ I
2 ; '
c.. 360 .6. .6. .56. (.6. (.6. moo =0 .66 060 .66 (2'00 who (.66 2100
O m
at» E . at)- F
‘E‘ZCO‘ . . o . _zw
g m-L.I . O . m.
j”. . o 8”-
E120- . . ' 13°“ .
I . ..
D a“ . . ”- o
40": ‘0' o .' . . .
'0... °. O o .
o 4. O O
“a shining?» 155013002100 0 :60 cbooéou'oorfifiooatoo
PAR (JAE m‘r') PAR 01: ur'r')

 

 

 

 

 

 

 

 

 

 

 

 

62

Figure 3: Response of gas exchange parameters of
micropropagated (MP) (A, C, E) and conventionally propagated
(CP) (B, D, F) 'Redchief' plants grown under full sun tx>
different levels of photosynthetically active radiation
(PAR). Gas exchange parameters: net photosynthesis (FWD
(A, B), mesophyll conductance (gm) (C, D) and stomatal
conductance (gs) (E, F). Different symbols represent

replications; 6 - 10 measurements per replication.

63

 

9L3

PM (mg 002 dur'hr")
7.5 i

f T

 

 

 

V
1500

V
1000 21m

 

gm(mmol 0"m")
9 4 as 9 a

$

 

 

1000' 2:00

 

3354

9: (mmol 0"m")
3 §

 

. 9

 

 

:2'00
PAR (pt m‘r’)

W
1500

 

=11

:04

 

 

 

fi'eéi'
3000001200

(5'00

(030 2100

 

9641193

i3

 

 

 

.?
' In.

I
1200

360060060

1
1:00

I
I” 2100

 

:00-
240-

"0-
120d
‘0. o‘
40..

 

 

:00 050 060 T230
PAROJEW)

t
1000

 

who 2:00

64

Figure 4: Response of net photosynthesis (mg C02 dm"2 hr’l)
to ambient C02 level in micropropagated (----) and
conventionally propagated 6—————) 'Earliglow' (A) and

'Redchief' (B) potted plants grown under full sun.

PN (mg COz dmflhr')

PN (mg CO2 dm‘hr")

65

 

 

   
 

 

 

420

 

 

   

  
 
 

 

 

25
MP 0
22.. CP 1 g .’
y..-0.061(x)-3.054 ' ”x
19" "30.953.5n» ””l . .
1 6- ‘ I’
l 3-
1 0-
7d
y,,n0.049(x)-3.229
4" FO.884"'
1- , i I
80 148 216 284 352
25 B I
. MP
22.. GP 0
y,,-0.062(x)-3.1 59
19' r-O.930“‘_' .
1 6-
1 3-
1 O-
7.1
yw-O.602(x)-3.640
4" r-O.936“'
1...... _
50 118 2i 6 284 362

C02 (PPFD)

420

66

Figure 5: Response of net photosynthesis (ug C02 gm dr wt'1
hr’l) to ambient C02 level 9 in micropropagated (----), and
conventionally propagated (—————) 'Earligltwv' (A) and

'Redchief' (B) potted plants grown under full sun.

 

PN (pg CO, mg dr wt"hr")

PN (ug 00. mg dr wt7‘hr")

67

 

 

   

 

 

 

    

 

4.0
0 MP
3.5- ° °"
300- N .
y -0.0076(x)-0.389
£0.96?" - ,x
2.5- ”,4” . '
200- I”” . .
o ’4
1.5-
1.0-
0.3- y -o.0052(x)—0.351
til-0.870;"
000 I l
60 146 216 264 352 420
4.0 B
0 MP
3.5- " °P .
300‘
y -0.0079 x -0.518
£0.809m0 . ,x’
2.5" ’.a’
’ o o
200- I
105‘
1.0-
~ 130.951";
0.0
80 1116 2i6 264 352 420

 

 

CO. (ppm) .

 

68

Figure 6: Response of mesophyll conductance (gs) on a

weight per leaf unit area basis to ambient C02 level in

 

micropropagated (----) and conventionally propagated ( ) 1
'Earliglow' (A) and 'Redchief' (B) potted plants grown

under full sun.

9, (mmol sr‘md)

9. (mmol 84m")

69

 

 

    

 

 

 

 

 

 

 

a A .1
0 MP
.. a GP y -0.0088(x)+2.434
7 130.535; ,
8" . . ‘a on"
5- ‘ 2-4"” ’ 0 '
4- . , “.;:‘ . I I . .
3..
2..
1.. , . . y,,-0.0067(x)+1.089
, r=0.511'
0 I ‘ 1
60 146 216 264 352 420
2..
. yc,-0.0097(x)+1.683
1.. a F0358“
I
O T i . I
80 148 216 264 352 420

00. (ppm)

 

70
carboxylation efficiencies were similar for MP and second
generation plants of both cultivars (Figure 4). However, on
a per dry weight basis, carboxylation efficiency was greater
(slopes significantly different p < 0.01) in E0 than in
E2 plants, although this difference was not seen in
'Redchief' (Figure 5). On a per dry weight basis, response
of 9m to increasing ambient C02 concentration was identical
in R0 and R2, but was maintained at a higher level in E0

plants compared to E2 (Figure 6).

Discussion

In general, micropropagated plants maintained higher gas
exchange rates than their field grown progeny. In a
previous study, it was suggested that higher rates of Pnet
observed in MP 'Earliglow' might be the result of increases
in fruit load (4). Data presented here demonstrate that
higher rates of Pnet exist even in deblossomed MP versus CP
plants. This indicates that leaf/fruit relationships are
not the primary cause of enhanced gas exchange responses in
micropropagated plants.

The two cultivars responded differently to plant
density. In MP 'Earliglow', increases in leaf density
resulted in concomitant increases in PN on a dry weight
basis, while in MP 'Redchief', PN was unchanged. In CP
plants of both cultivars, PN decreased as leaf density

increased. The difference in response between the two

71

cultivars may relate to factors influencing mesophyll
conductance and carboxylation efficiency.

Mesophyll conductance (gm) has been described as the
C02 diffusional pathlength to sites of carboxylation (20).
A strong correlation between weight per leaf unit area and
mesophyll surface area has been documented in strawberry
(7,13). The ratio of mesophyll surface area to leaf surface
area contributes to photosynthetic capacity of leaves (18);
leaves with higher ratios have higher photosynthetic
capacity. In CP plants, weight per leaf unit area was not
significantly correlated with density and PN dropped as
density increased. In leaves of MP 'Earliglow', large
decreases in weight per leaf unit area were observed
as density increased; these were accompanied by increases in
PN.* In MP 'Redchief', weight per leaf unit area decreased
with increasing density, and rates of PN were unchanged.
Perhaps MP plants are more plastic than CP plants and cxn1
maintain photosynthetic rates across a broader range of
conditions by regulating mesophyll surface area.

Carboxylation efficiency was greater in MP than in CP
'Earliglow', but this was not true in 'Redchief' (Fig.
5). Response of mesophyll conductance to increasing C02
concentration on a per dry weight basis did not differ in
R0 and R2 plants, but declined with propagation generation
in 'Earliglow' (Fig. 6). A number of factors could

influence carboxylation efficiency including levels or

72
activity of ribulose-l, 5-bisphosphate carboxylase/oxygenase
and regeneration of its substrate, photorespiration, and
triose phosphate utilization (20). Differences in
carboxylation behavior could account for the higher rates of
Pnet in MP 'Earliglow' than in MP 'Redchief' observed in
a previous study (4).

In summary, micropropagated strawberry cultivars produce
more stolons than conventionally propagated plants
(8,15,21). Micropropagation apparently increases yield only
in genotypes which are well adapted to high plant densities.
Path analysis indicates that MP cultivars which do not
exhibit increased yields are sensitive to crowding stress as
CP plants (Cameron and Hancock, unpublished data). The
ability of some genotypes to increase reproductive effort
(11) and photosynthetic rate (4) with increasing plant
density plays a role in allowing plants to adjust to a more
competitive environment.

The enhanced vigor associated with micropropagated
strawberry plants does appear to dissipate with subsequent
generations of conventional propagation, but the first
generation progeny of MP plants maintain an enhanced level
of vigor over CP plants (3) sufficient to increase
productivity in the first bearing year (5). For this
reason, MP plants are of benefit not only to the nurseryman,

but also to the grower.

73

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1.

10.

11.

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Barden, J.A. 1974. Net photosynthesis, dark respiration,
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Cameron, J.S. and J.F. Hancock. 1986. Enhanced vigor in
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Cameron, J.S., J.F. Hancock and J.A. Flore. 1986.
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Cameron, J.S., J.F. Hancock and T.M. Nourse. 1985. The
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Chabot, B.F. 1978. Environmental influences on
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Chabot, B.F. and J.F. Chabot. 1977. Effects of light and
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Damiano, C., 1980. Strawberry micropropagation. p.11-22,
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Davies, F.s. and J.A. Flore. 1986. Gas exchange and
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Guttridge, C.G. and H.M. Anderson. 1973. The
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Hancock, J.F, J.W. Moon and J.A. Flore. 1984. Within-row
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74

Hull, J., J. Moulton and J. Flore. 1977. Growing
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Jurik, T.W., J.F. Chabot and B.F. Chabot. 1979. Ontogeny
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Kappel, F. and J.A. Flore. 1983. Effect of shade on
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Marcotrigiano, M., H.J. Swartz, S.E. Gray, D. Tokarcik
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performance of tissue-culture propagated strawberry
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Moon.;n:., J.W. and J.A. Flore. 1986. A BASIC computer
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Moran, 12., 1982. Formulae for determination of
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n,n-dimethylformamide. Plant Physiol. 69:1376-1381.

Nobel, P.S., L.J. Zaragoza and W.K. Smith. 1975.
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rate, and illumination level during development for
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Physiol. 55:1067- 1070. .

 

Sams, C.E. and J.A. Flore. 1982. The influence of age,
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Sharkey, T.D., 1985. Photosynthesis in intact leaves of

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Swartz, H.J., G.J. Galletta, and R.H. Zimmerman. 1981.
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