THE EVALUATION OF Cuphea pulchra AND Cuphea schumannii AS POTENTIAL NEW ORNAMENTAL CROPS FOR INTRODUCTION INTO THE FLORICULTURE INDUSTRY
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
The Degree Doctor of Philosophy in the
Graduate School of The Ohio State University
By
Jennifer Hrach Leopold, B.S.
*****
The Ohio State University 2004
Dissertation Committee: Approved by Dr. James Metzger, Advisor
Dr. Margaret McMahon ______Advisor Dr. David Gardner Horticulture and Crop Science Dr. Michael Boehm Graduate Program
ABSTRACT
Two species, Cuphea pulchra and Cuphea schumannii, native to Brazil and
Mexico respectively, were evaluated for their potential as new ornamental crops. Both species are characteristic of the genus as suffrutescent sub-shrubs bearing intensely colored orange, ribbed flowers. No information exists regarding the horticultural qualities of either species. The objectives of the current research were to evaluate the species for ornamental qualities and begin developing information pertinent to commercial production.
Propagation by vegetative cutting was the most efficacious method of propagating both species. Use of a 0.1% indol-3-butyric acid rooting hormone promoted increased root development on C. schumannii stem cuttings but had no significant effect on C. pulchra cuttings. Initial greenhouse observations and the results of three consecutive years of outdoor trials demonstrated that C. schumannii possess relatively little horticultural potential, due to an indeterminate growth habit, high susceptibility to green peach aphid infestation and development of leaf intumescence under greenhouse conditions. Conversely, C. pulchra holds much promise as an annual, landscape/garden bedding plant with its compact growth habit, sustained heavy production of attractive flowers, and resistance to greenhouse and outdoor insect pests.
ii Continued studies of the species evaluated methods of growth regulation, production temperature, and photoperiodic requirements for flowering. Evaluation of plants treated with chemical growth retardants daminozide, paclobutrazol, and chlormequat indicated that growth of C. schumannii can be effectively and uniformly
controlled with a concentration of 5000 ppm daminozide. Evaluation of C. pulchra
showed that the plant height and form can be most successfully controlled by removing
the plant apex. Results of controlled environment growth chamber studies demonstrated
that plants of both species were of higher quality and flowered most readily when
maintained under a day/night temperature regime of 21ºC/18ºC in comparison to plants
grown under a 13ºC/10ºC or 29ºC/26ºC temperature regime. A study evaluating the
photoperiodic response of both species revealed that C. schumannii can be categorized as
a day-neutral or a very weak facultative long-day plant, while C. pulchra is a facultative
long-day plant.
Preliminary investigations were performed to determine the nature of insect
resistance in C. pulchra. Choice and no-choice settling assays were used to evaluate
resistance to green peach aphid and revealed that resistance is due in large part (if not all)
to the presence of glandular trichomes on the stem and flower surfaces. Preliminary
analysis by thin layer chromatography showed that the main component of the trichome
exudate was a compound(s) that co-chromatographed with acyl esters of sucrose. Acyl
esters of glucose and sucrose have been shown to be the primary compounds responsible
for plant resistance to insect herbivores of wild species of tomato, potato, and tobacco.
iii Dedicated in Memory of Elaine R. Hrach, Ph.D.
iv ACKNOWLEDGMENTS
I take this opportunity to express my heartfelt gratitude to all of those who, over the last five years, have contributed to the success of this research.
I wish to thank my advisor, James Metzger, for his guidance, willingness to share his wealth of knowledge, and for allowing me the opportunity to learn from my own mistakes and successes as a researcher.
I thank Peg McMahon for her encouraging words and readiness to be an advisor, mentor, and role model.
I also thank members of my dissertation committee, Dave Gardner who encouraged with his words “be proud of what you do” and Mike Boehm who was willing to serve as a committee member on short notice.
Finally, I am most grateful to my parents Joe and Peg Hrach, and my wonderful husband Chris for their love and support of all that I do.
v VITA
May 29, 1977……………………………… Born – New Castle, Pennsylvania, U.S.A.
1999……………………………………….. B.S. Biology, Allegheny College
1999 – present…………………………….. Graduate Research and Teaching Associate,
The Ohio State University
PUBLICATIONS
1. Leopold, J.H., J.D. Metzger, and S.A. Graham. 2003. Development and evaluation of Cuphea pulchra and Cuphea schumannii as new landscape ornamentals. ISHS ACTA Horiculturae. 624: 85-91.
FIELD OF STUDY
Major Field: Horticulture and Crop Science
vi TABLE OF CONTENTS
Page
Abstract…………………………………………………………………………….. ii
Dedication...... iv
Acknowledgments...... v
Vita…………………………………………………………………………………. vi
List of Tables………………………………………………………………………. x
List of Figures……………………………………………………………………….xv
Chapters:
1. Review of literature………………………………………………………... 1
1.1. U.S. floriculture industry……………………………………………1
1.2. New crop research…………………………………………………..2 1.3. Evaluation of new floriculture crops………………………………..4 1.4. Cuphea……………………………………………………………... 7 1.4.1. Cuphea pulchra……………………………………………..9 1.4.2. Cuphea schumannii………………………………………....11 1.5. Issues surrounding plant acquisition………………………………..12 1.5.1. Bioprospecting……………………………………………...13 1.5.2. Convention on biological diversity…………………………13 1.5.3. Access and benefit sharing………………………………… 14 1.5.4. NBI-Ball agreement: a working example………………….. 15 1.6. Production research for a type III species………………………….. 16 1.6.1. Propagation………………………………………………… 16 1.6.2. Control of flowering……………………………………….. 19 1.6.3. Growth regulation………………………………………….. 22 1.6.4. Temperature………………………………………………... 25 1.6.5. Plant-insect interactions……………………………………. 27 1.7. Research objectives………………………………………………....31
vii 2. Initial observation of performance under greenhouse and simulated landscape conditions………………………………………... 32
2.1. Introduction…………………………………………………………32 2.2. Materials and methods……………………………………………... 34 2.2.1. Initial greenhouse observations……………………………..34 2.2.2. Outdoor Trials……………………………………………… 36 2.3 Results and discussion……………………………………………... 42 2.3.1 Initial greenhouse observations……………………………..42 2.3.2 Outdoor trials………………………………………………. 54 2.4 Conclusions………………………………………………………… 63
3. Propagative methods……………………………………………………….. 64
3.1. Introduction………………………………………………………… 64 3.2. Materials and methods……………………………………………... 66 3.2.1. Germination rates of untreated seed……………………….. 66 3.2.2. Seed embryo excision……………………………………… 67 3.2.3. Acceleration of cutting rooting with hormone treatment…... 69 3.3. Results and discussion……………………………………………... 70 3.3.1. Seed germination…………………………………………... 70 3.3.2. Seed embryo excision……………………………………… 73 3.3.3. Effect of rooting hormone on root development…………… 75 3.3.4. Effect of Hormodin®-1 on root development……………... 77 3.4. Conclusions………………………………………………………… 79
4. Preliminary investigation of the effects of temperature on growth and development…………………………………………………………… 80
4.1. Introduction………………………………………………………… 80 4.2. Materials and methods……………………………………………... 83 4.3. Results and discussion……………………………………………... 85 4.4. Conclusion…………………………………………………………. 93
5. Methods of growth regulation……………………………………………… 95
5.1. Introduction………………………………………………………… 95 5.2. Materials and methods……………………………………………... 99 5.2.1. Chemical growth retardants………………………………... 99 5.2.2. Photoselective filters………………………………………..103
viii 5.3. Results and discussion……………………………………………... 106 5.3.1. Chemical growth regulation………………………………... 106 5.3.2. Non-chemical growth regulation by photoselective filters… 125 5.4 Conclusions………………………………………………………… 128
6. Photoperiodic effects on flowering and growth……………………………. 130 6.1. Introduction………………………………………………………… 130 6.2. Materials and methods……………………………………………... 132 6.3. Results and discussion……………………………………………... 136 6.3.1. Cuphea schumannii…………………………………………136 6.3.2. Cuphea pulchra…………………………………………….. 141 6.4. Conclusions………………………………………………………… 146
7. Role of trichomes and trichome exudate in the resistance of Cuphea pulchra to green peach aphid……………………………………... 148
7.1. Introduction………………………………………………………… 148 7.2. Materials and methods……………………………………………... 154 7.2.1. Identification of trichome types……………………………. 154 7.2.2. Whole plant settling assay…………………………………. 154 7.2.3. Adult green peach aphid preference assay…………………. 157 7.2.4. Analysis of C. pulchra glandular trichome exudates………. 160 7.3. Results and discussion……………………………………………... 161 7.3.1. Identification of trichome types by SEM…………………... 161 7.3.2. Verification of deterrence by choice/ no-choice settling assay……………………………………………….. 165 7.3.3. Influence of glandular trichome exudate on green peach aphid settling…………………………………..170 7.3.4. Analysis of C. pulchra glandular trichome exudate……….. 173 7.4. Conclusions………………………………………………………… 175
8. General Conclusions………………………………………………………...178
References………………………………………………………………………….. 182
ix LIST OF TABLES
Table Page
2.1 2001 Trial climate data. Average maximum and minimum air temperature and total rainfall for June through November 2001…………... 37
2.2 2002 Trial climate data. Average, maximum, and minimum air temperature and total rainfall for May through November 2002………….. 39
2.3 2003 Trial climate data. Average, maximum, and minimum air temperature and total rainfall for May through November 2003…………... 41
2.4 Monthly cumulative PFF exposure of C. pulchra and C. schumannii trial plants under full sun conditions for June through October 2003…….. 41
2.5 Effects of soil moisture on development of leaf intumescence and other growth parameters of Cuphea schumannii (Trial 1)…………………. 52
2.6 Effects of soil moisture on development of leaf intumescence and other growth parameters of Cuphea schumannii (Trial 2)…………………. 53
3.1 Percent germination of Cuphea schumannii and Cuphea pulchra seed 7, 14, and 21 days after sowing. ……………………………………………72
3.2 Effect of seed embryo excision on mean percent germination of Cuphea pulchra and Cuphea schumannii…………………………………………... 74
3.3 Effects of exogenous rooting hormone (Hormodin®-1) on the average root number and average length of roots formed on vegetative cuttings of Cuphea schumannii and Cuphea pulchra at 7, 11, 14, 18, and 21 days after sticking………………………………………………………………...78
4.1 Cumulative photosynthetic photon flux, average hourly temperature, and average hourly relative humidity environmental data collected from growth chambers maintaining the experimental temperature regimes of 13ºC/10ºC (low), 21ºC/18ºC (mid), and 29ºC/26ºC (high)…………….. 84
x 4.2 Treatment effects on pre-harvest parameters of Cuphea pulchra plants maintained under 13ºC/10ºC (low), 21ºC/18ºC (mid), or 29ºC/26ºC (high) growth chamber temperature regimes…………………... 87
4.3 Treatment effects on pre-harvest parameters of Cuphea schumannii plants maintained under 13ºC/10ºC (low), 21ºC/18ºC (mid), or 29ºC/26ºC (high) growth chamber temperature regimes…………………... 88
4.4 Treatment effects on post-harvest parameters of Cuphea pulchra plants maintained under 13ºC/10ºC (low), 21ºC/18ºC (mid), or 29ºC/26ºC (high) growth chamber temperature regimes……………………………………… 89
4.5 Treatment effects on post-harvest parameters of Cuphea schumannii plants maintained under 13ºC/10ºC (low), 21ºC/18ºC (mid), or 29ºC/26ºC (high) growth chamber temperature regime………………………………………. 89
5.1 Cumulative photosynthetic photon flux, average temperature, and average relative humidity of greenhouse growing conditions for the experimental periods of 5 and 8 weeks for C. schumannii and C. pulchra respectively………………………………………………………………… 100
5.2 Cumulative photosynthetic photon flux for photoselective filter trial 1 (four week period March 27, 2002- April 24, 2002) and cumulative PFF and average hourly temperature for trial 2 (four week period July 22, 2002 – August 19, 2002)……………………………………………………………105
5.3 Effect of Cycocel concentrations of 800, 1250, and 2000 ppm on Cuphea schumannii plant growth parameters………………………………………. 107
5.4 Effect of B-Nine concentrations of 2500, 3750, and 5000 ppm on Cuphea schumannii growth parameters…………………………………………….. 108
5.5 Effect of Bonzi concentrations 10, 25, and 24 ppm on Cuphea schumannii plant growth parameters……………………………………………………. 110
5.6 Effect of apex removal (pinch) on Cuphea schumannii plant growth parameters………………………………………………………………….. 111
5.7 Evaluation summary for CGR treated Cuphea schumannii………………... 114
5.8 Effect of Cycocel concentration 800, 1250, and 2000 on Cuphea pulchra growth parameters………………………………………………………….. 117
5.9 Effect of B-Nine concentrations of 2500, 2750, and 5000 ppm on Cuphea pulchra growth parameters………………………………………... 119
xi
5.10 Effect of Bonzi concentrations 10, 25, and 24 ppm on Cuphea pulchra plant growth parameters……………………………………………………120
5.11 Effect of apex removal (pinch) on Cuphea pulchra plant growth Parameters…………………………………………………………………. 121
5.12 Evaluation summary for PGR treated Cuphea pulchra……………………. 123
5.13 Comparative results of Cuphea pulchra plants grown under a far-red reducing filer, neutral density, or open bench (no filter) conditions………..126
5.14 Comparative results of Cuphea schumannii plants grown under far-red reducing filter, neutral density, or open bench (no filter) conditions……… 127
6.1 Average hourly PPF, average daily PFF, average temperature (ºC) and average relative humidity (%) of the growth chambers maintaining an 8 hr (SD) or 8 hr + 8 hr extension (LD) photoperiod.……………………………133
6.2 Comparison of plant growth parameters of Cuphea schumannii maintained for 7 weeks under short-day (SD) or long-day (LD) photoperiodic conditions…………………………………………………………………... 140
6.3 Comparison of the average number of buds and flowers visible on C. pulchra plants held under short-days (SD) or long-days (LD) at 3, 5, 7, 9, and 11 weeks after experiment initiation……………………………... 142
6.4 Comparison of the average number of buds and flowers visible on C. pulchra plants under short-days (SD) or plants that had been transferred from short-days to long-days at 1, 3, and 6 weeks after plant transfer to LD………………………………………………………… 143
6.5 Plant growth parameters of Cuphea pulchra plants maintained under long-days (LD), transferred from short-days (SD) to long days (LD), and maintained under short-days (SD)…………………………………………..145
7.1 Defining characteristics and plant surface locations of the seven types (and sub-types) of trichomes on Cuphea plant surfaces as identified in Amarasinghe et al. (1991) in a survey of 136 species in the genus………... 149
7.2 Effect on Cuphea pulchra and Cuphea schumannii growth parameters of plants maintained under choice or no-choice aphid settling conditions…169
7.3 Mean number of aphids on C. pulchra cuttings washed with EtOH to remove trichome exudates droplets and untreated cuttings...... 172
xii LIST OF FIGURES
Figure Page
1.1 Condensed adaptation of the overall scheme of a new crops evaluation system as described by Roh and Lawson (1988)…………………………...5
1.2 Map of Bahia and Minas Gerais, Brazil showing the distribution of populations of Cuphea pulchra ……………………………………………..9
1.3 Cuphea pulchra flower.………………………………………………...... 10
1.4 Map of Mexico showing the distribution of populations of Cuphea schumannii.…………………………………………………………………11
1.5 Cuphea schumannii flower…………………………………….……………12
2.1 Cuphea schumannii maintains a “leggy” and indeterminate growth habit making it unsuitable for production as a potted crop…………………43
2.2 A. Cuphea pulchra line “A” maintains a rounded, compact growth habit. B. Stems and leaves of C. pulchra “A” are soft and flexible which give the plant a delicate appearance………………………………… 45
2.3 A. Cuphea pulchra line “B” maintains an upright or spreading yet more open and free form than C. pulchra “A”. B. Stems and branches are thick and leaves are stiff, giving the plant a rigid appearance…………. 45
2.4 A. Cuphea pulchra line “C” maintains a free branching, sprawling growth habit. B. Similar to line “B”, branching and leaves are stiff, however, leaves have finely scalloped margins……………………………. 46
2.5 A. Cuphea pulchra “white” is a sport of line “A” maintaining the same rounded, compact, and delicate growth habit as its parent. B. Foliage of “white” is a deeper green than its parent plant and the floral tubes are whitish to light pink color…………………………………. 46
xv 2.6 A. Lateral cutaway view of inner floral tube of Cuphea pulchra. B. Lateral view of the mouth of the floral tube of C. pulchra...... 47
2.7 Cuphea schumannii susceptibility to green peach aphid (Myzus persicae) infestation under greenhouse conditions..…………………………………..48
2.8 Development of intumescence injury on the foliage of Cuphea schumannii under controlled environmental conditions of the greenhouse...50
2.9 A. Bumblebee on Cuphea pulchra flower. B. Honeybee and yellow jacket wasp on Cuphea schumannii flower.…………………………...... 55
2.10 Nectar robbing from flowers of C. schumannii. Bees were capable of making an incision through rear dorsal portion of the floral tube (indicated by arrow) to gain direct access to the nectaries……………….. 56
2.11 Cuphea pulchra lines “A”, “B”, and “C” height data collected during the 2002 summer trial from June 7, 2002 through October 2, 2002……….. 58
2.12 Cuphea pulchra lines “A”, “B”, and “C” plant diameter data collected during the 2002 summer trial from June 7, 2002 through October 2, 2002.. 59
2.13 Cuphea schumannii plant height data collected during the 2002 summer trial from June 7, 2002 through October 2, 2002…………………………... 59
2.14 Cuphea schumannii plant diameter data collected during the 2002 summer trial from June 7, 2002 through October 2, 2002…………………………... 60
2.15 Cuphea pulchra lines “A”, “B”, “C”, and “white” height data collected during the 2003 summer trial from May 28, 2003 through October 8, 2003………………………………………………………………………... 61
2.16 Cuphea pulchra lines “A”, “B”, “C” and “white” plant diameter data collected during the 2003 summer trial from May 28, 2003 through October 8, 2003……………………………………………………………. 61
2.17 Cuphea schumannii plant height data collected during the 2003 summer trial from May 28, 2003 through October 8, 2003…………………………. 62
2.18 Cuphea schumannii plant diameter data collected during the 2003 summer trail from May 28, 2003 through October 8, 2003…………………………. 62
3.1 Seed hairs of a Cuphea schumannii seed. Hairs move out of the epidermal cells of the seed coat of Cuphea species after a short duration of exposure to water. The hair serve to anchor the seed to the germination substrate…. 71
xv 3.2 Images of Cuphea pulchra seed with seed coat (left) and excised from seed coat (right)……………………………………………………………..74
3.3 Root formation on Cuphea schumannii vegetative cuttings treated with (from left to right) Hormodin®-1, Hormodin®-3, Rootone®F, and the untreated control 19 days after sticking………………………………………………. 76
4.1 Effect of temperature on growth and development of Cuphea pulchra…….90
4.2 Low temperature effects on anthocyanin production in foliage of C. pulchra……………………………………………………………………...91
4.3 Effect of temperature on growth and development of Cuphea schumannii…92
5.1 Experimental chambers used to evaluate plant growth under far-red absorbing films……………………………………………………………...104
5.2 Cuphea schumannii treated with Bonzi® (paclobutrazol) plant growth regulator…………………………………………………………………….112
5.3 Pinched C. schumannii (right) vs. control (left)…………………………… 112
5.4 Cuphea schumannii treated with 5000 ppm daminozide (B-Nine®) was chosen by evaluators to have the best overall appearance when compared with untreated controls, pinched plants, and those that had been treated with other concentrations of commercially available CGRs…………………………... 115
5.5 Cuphea pulchra treated with 800, 1250, and 2000 ppm (left to right) of Cycocel® PGR……………………………………………………………….116
5.6 Cuphea pulchra treated with 10, 25, and 40ppm (groups left to right) Bonzi®……………………………………………………………………….122
5.7 Control (left) vs. pinched (right) Cuphea pulchra………………………….124
6.1 Graphical depiction of the difference in the number of visible buds and flowers produced on Cuphea schumannii under short-day (SD) and long-day (LD) photoperiods at 3, 5, and 7 weeks after experiment initiation……………... 137
6.2 Digital image comparing the morphological differences between C. schumannii maintained under short day (right) and long day (left) photoperiod for 7 weeks…………………………………………………… 138
xvi 6.3 Digital image comparing the morphological difference of C. pulchra plants that had been maintained under a long-day (left) and a short-day (right) photoperiod for 11 weeks…………………………………………...143
7.1 Wood frame cages constructed for the whole plant aphid settling assay conducted under growth chamber conditions………………………………157
7.2 Cages designed for the aphid settling preference assay…………………….158
7.3 Scanning electron microscope image of the interior floral tube surface of Cuphea schumannii………………………………………………………162
7.4 Scanning electron microscope image of the stem surface of Cuphea schumannii…………………………………………………………………..163
7.5 Scanning electron microscope image of the exterior floral tube surface of Cuphea pulchra…..………………………………………………………164
7.6 Close-up scanning electron microscope image of a multicellular resin- secreting glandular trichome (TYPE 1) on the stem surface of Cuphea pulchra……………………………………………………………………... 165
7.7 Comparison of mean number of total aphids settled per Cuphea pulchra or Cuphea schumannii plant under green peach aphid choice settling conditions………………………………………………………………….. 166
7.8 Comparison of mean number of total aphids settled per Cuphea pulchra or Cuphea schumannii plant under green peach aphid no-choice settling conditions…………………………………………………………………... 167
7.9 Mean number of immature aphids on C. pulchra and C. schumannii plants under choice or no-choice treatment conditions. The significantly higher number of immature aphids on C. schumannii reflects the higher aphid fecundity on this species over C. pulchra………………………………….. 168
7.10 Mean number of aphids on stem and leaves of untreated C. pulchra cuttings and cuttings treated by an ethanol wash to remove trichome exudates droplets…………………………………………………………… 172
7.11 Digital photograph of the developed TLC plate comparing the banding pattern produced by the sucrose octanoate standard (column 1), a 1:10 dilution of the sucrose octanoate standard (column 2), and the stem wash sample of C. pulchra (column 3)…………………………………………... 174
xvii CHAPTER 1
REVIEW OF LITERATURE
1.1 U.S. Floriculture Industry
In recent years gardening has become the number one leisure activity among
Americans (National Gardening, www.garden.org). This growth in consumer interest in
ornamental horticulture has led to a recent and rapid expansion of the floriculture
industry. The floriculture industry continues to make a significant contribution to the
U.S. economy.
Floriculture crops include bedding plants, flowering plants, foliage plants, cut
cultivated greens, cut flowers, and propagative material. The total wholesale value of
floriculture crops grown in the U.S. by operations exceeding the $10,000 sales level in
2003 reached $5.07 billion (USDA, 2004). California is the leading producer with just
over $1.00 billion, followed by Florida, Michigan, Texas, and New York, respectively
(USDA, 2004). In 2003, Ohio ranked seventh overall with $181.5 million in wholesale
floriculture items produced (USDA, 2004).
In 2003, as estimated by the Economic Research Service (ERS), bedding and
garden plants accounted for 51 percent of production of finished floral crops. Bedding
and garden plants wholesale value, at $2.42 billion in 2003, was the largest contributor to
the value of production, up 1 percent from 2002 (USDA, 2004). Although commercial
1 Producers of bedding and garden plants are located throughout the United States, five states, California, Michigan, Texas, Ohio, and Florida account for 41 percent of the total bedding and garden plant value.
1.2 New Crop Research
Product novelty is an important attribute that makes the ornamental industry unique among agricultural industries (Halevy, 1999). Consumers seek out the “new and different” in clothing styles and home décor and should be no surprise that this trend continues when it comes to plant material for landscapes and gardens. Consumers fuel the floriculture industry by placing the demands for new and better landscape ornamentals. There will always be the standard varieties of impatiens, geraniums, and petunias that adorn the front yards, containers, and hanging baskets of American households, but novelty is very important.
Why is there a continual need for research of new ornamental plants? The reasons are multifold, however, there are several incentives that drive the development of new crops (L. Hatch, 1998) . For the grower, new ornamental plant research and introduction can provide better pest and disease resistance, removal of unfavorable growth traits, uniform characteristics and cost-effective propagation. The advantages of new ornamental plants extend to the consumer through greater variety of plants, plants that are more tolerant of environmental stresses, and plants that adapt to the changing human spaces.
There is some debate as to what actually constitutes a new floriculture crop. A
“new” floriculture crop can be a newly discovered genera or species; newly generated
2 cultural information for cultivars of plants grown in earlier years; plants that are
cultivated in foreign countries but have not been introduced in the United States; or crops
that can be produced with new production technologies that enhance crop quality and/or
shorten total production time (Roh and Lawson, 1988). Given this broad definition,
thousands of new floriculture crops are available for study.
Growers and university researchers are conducting additional new crops programs
and research independently or in cooperation with USDA efforts (Roh and Lawson,
1996). A significant university-based new crops program is being conducted at the
University of Georgia under the direction of Allan M. Armitage. Armitage (1986, 1987)
outlines in detail three main areas of new floriculture crops research that have evolved
due to increasing efforts toward new crop research. The first area of research is the
evaluation of new plant selections or clones of well established, or type I, ornamental species with a high degree of prior selection (Armitage, 1986, 1987). The second area of new crop research deals with new uses for well-known minor crop, or type II species
(Armitage, 1986; Armitage, 1987). An example would be evaluation of the potential of a traditional bedding plant to perform and be marketed successfully as a new pot crop.
The third area of new crop research involves the development and evaluation of a species for which little or no garden or greenhouse cultural information exists (Armitage, 1986,
1987). Type III species typically have no prior selection and little or no information is known concerning flowering physiology or performance under production conditions. Of course, type I, type II, and type III species categorization is dynamic as research continues.
3 The potential for basic research is rich for type III species. Because little is
known about control of flowering in type III species, photoperiod manipulation and experiments on factors affecting the onset of flowering such as temperature and water status are of high significance. However, information pertinent to commercial use, such as propagation techniques, height control, optimum media pH, irradiance levels and optimum temperatures must also be fully explored and established for a species before acceptance into the industry.
1.3 Evaluation of New Floriculture Crops
More plant species possessing potential as floriculture crops exist than can possibly be fully evaluated through a research program. The task of the researcher is to recognize the potential success of a species while at the same time discarding those species with little chance of acceptance in the industry (Armitage, 1988). Therefore, it is important that a new crops program maintain a systematic approach to evaluation.
Roh and Lawson (1988) provide a flowchart scheme of a new crops evaluation system to illustrate a systematic approach to evaluation (Figure 1.1). Introduction of a new floriculture crop begins with acquisition of both foreign and domestic species possessing different and interesting characteristics.
Once plant material has been selected for evaluation, it undergoes an initial screening process, which assesses the ease of propagation, aesthetic qualities, flowering time, and insect and disease susceptibility. This stage allows researchers to identify plant material that has little potential and should be discarded from the program as well as material that may require more extensive study.
4
Collection
Initial Observation exclude
Easy Difficult Propagation Propagation
Extensive Evaluation exclude
Cultural Information
University Nationwide Evaluation Industry
Official Introduction
Figure 1.1. Condensed adaptation of the overall scheme of a new crops evaluation system as described by Roh and Lawson (1988).
If propagation of the plant material is considered commercially feasible and it passes standards of initial observation then aspects of production can be addressed. 5 Production issues include media and nutrition requirements, growth regulation,
photoperiodic requirements, and temperature effects on growth and development. This research will form the backbone of the information provided to commercial growers
(Armitage, 1986). Based on performance during this period of extensive investigation, researchers may again make the decision to remove plant material from the program.
Upon development of an initial production protocol for a particular crop, the cultural information is released at trade meetings and through publications. Evaluation of this protocol at various other locations and environmental conditions is vital for the success of the total introduction program (Roh and Lawson, 1988). Based on performance under different production conditions, recommendations can be made as to where and how the new crop species would be best produced. The time necessary for a regional and/or nationwide production trial is dependent on crop and the information about it that has yet to be developed. A maximum of five years is projected for a plant that is relatively new or has not been extensively investigated (Roh and Lawson, 1988).
For plants that have had limited commercialization or have been previously introduced in other countries the period is reduced to a maximum of two to three years (Roh and
Lawson, 1988). For a large company like Ball FloraPlant which introduces 80-100 vegetatively propagated plant lines per year (including line extensions, upgrades, improvements and new classes) the average time between the acquisition of plant material and commercial introduction ranges from four to seven years (E. Hunt, personal communication). This period is dependent on the crop yield (for seed propagated crops) and how quickly it grows as well as the extent of evaluation being conducted.
6 The final phase of new floriculture crop introduction is the marketing of the crop.
At this point it is critical to the success of the crop that growers, wholesalers, and retailers are all aware of and familiar with the cultural information of the plant. If the consumer is not properly informed by these sources about the necessary care and handling of the plant, the disappointment of poor performance may restrict the economic potential.
1.4 Cuphea
Cuphea is the largest of the 31 genera of the family Lythraceae, encompassing about 260 species that range in natural habitat from temperate to tropical climates of eastern U.S. to southern Argentina (Graham, 1988; Graham 1998). The genus is comprised largely of perennial herbs and small shrubs that produce ribbed tubular flowers that are often intensely colored. Characteristics such as leaf morphology, stem morphology, and inflorescence arrangement tend to be specific to the section to which a particular species belongs.
Primary interest in the genus was for its potential as an oilseed crop. Cuphea seed is a source of medium chain fatty acids, including lauric acid (Graham, 1988). To reduce
U.S. dependence on imported coconut and palm kernel oils as primary sources of lauric acid for manufacturing soap, detergents, and surfactants, the USDA-ARS in Phoenix, AZ initiated research in 1983 to domesticate species of Cuphea for potential production as a new arid-land crop (Thompson, 1985). Most Cuphea species exhibit characteristics of undomesticated species, such as seed shattering, indeterminate patterns of growth and flowering, and sticky glandular hairs on stems leaves, and/or flowers, which limit
7 agronomic potential. The glandular hairs are of particular concern because the viscous, resinous exudates clog machinery, hindering seed harvest (Hisinger and Knowles, 1984).
Some Cuphea species possess considerable potential for use as landscape and garden ornamentals. Selections of species such as C. glutinosa, C. hyssopifolia, C. ignea, and C. llavea have already been developed for use as garden and landscape ornamentals for the southeastern US (Jaworski and Phatak, 1991: Jaworski and Phatak, 1991;
Jaworski and Phatak, 1992). One species, C. llavea, has been developed as a potential annual bedding plant in the northeastern U.S. No literature has been found to suggest that any serious efforts have been made to evaluate Cuphea species as perennial or annual landscape ornamentals in Midwestern regions. Similarly, no literature exists to suggest that any efforts have been made to evaluate species C. pulchra or C. schumannii for ornamental potential.
The two species under investigation in this research: C. pulchra and C. schumannii are both species in the section Melvilla Koehne. The first complete taxonomic treatment of the section was conducted by Koehne (1881-1883) when composing a monograph of the genus Cuphea (Graham, 1988). The original taxonomy of the section included 27 species arranged in six subsections (Graham, 1990). After studies of recent collections, the section Melvilla is now comprised of 47 species and an updated monographic key has been provided to facilitate the large number of Cuphea specimens gathered (Graham, 1990).
S.A. Graham (1990) describes the species of the section Melvilla as being the most striking of the large neotropical genus Cuphea. Species of the section are characterized by thick, dorsally convex hypanthia (floral tube) that are commonly 15 to
8 33 mm long and frequently deep red to yellow in color. Representatives of the section
are distributed from northern Mexico to Argentina in a variety of habitats.
1.4.1 Cuphea pulchra
Cuphea pulchra Moric is a perennial native to the states of Bahia and Minas
Gerais, Brazil (Figure 1.2). It can be found in a range of locations from cut over
Figure 1.2. Map of Bahia and Minas Gerais, Brazil showing the distribution of populations (indicated by black dots) of Cuphea pulchra, which is native to these two states only. (This map was first published in Systematic Botany Monographs and is being reprinted with the expressed written consent of the author, S.A. Graham) 9 woodland to closed wood cerrado, among rocks and on sandstone at elevations of 500-
1400 m (S.A. Graham, personal communication). In its native habitat, C. pulchra
flowers nearly all year but most abundantly in January, February, March, June, July,
August, September, October, November, and December (S.A. Graham personal
communication). This species is shrub-like, producing erect stems, but maintaining a
compact habit. Branching is dense with dark green, ovate leaves. Flowers are three cm.
long orange tubes lacking petals and are clustered at the branch terminals (Figure 1.3).
Figure 1.3. Cuphea pulchra. The bright orange flowers of the species are approximately three cm long tubs that lack petals and are clustered at branch terminals.
10
1.4.2 Cuphea schumannii
C. schumannii is native to a narrow north-south distribution from Vera Cruz to
Oaxaca, Mexico (Figure 1.4). It is a sub-shrub perennial found in disturbed secondary
Figure 1.4. Map of Mexico showing the distribution of populations (indicated by open circles) of Cuphea schumannii, which is native to a north-south distribution from states of Vera Cruz to Oaxaca. Dark circles represent the distribution of another species Cuphea salvadorensis. (This map was first published in Systematic Botany Monographs and is being reprinted with the expressed written consent of the author, S.A. Graham)
11 mesophytic woods, in sunny, open, moist roadside ditches, and among shade trees in coffee plantations at elevations of 750-1300 m (S.A. Graham personal communication).
In its native habitat, C. schumannii flowers from late November through July. This species is shrub-like, producing tall erect stems bearing large ovate leaves. C. schumannii produces axillary flowers with a vibrant three cm long orange floral tube and two purple petals located dorsally at the mouth of the floral tube (Figure 1.5).
Figure 1.5. Cuphea schumannii. The species produces three cm, orange tubular flowers with two small purple petals located dorsally at the mouth of the floral tube.
1.5 Issues Surrounding Plant Acquisition
The first step in a new crops research program is the acquisition of plant material.
Such material may come from domestic and foreign sources. Significant and 12 controversial issues arise when it comes to attaining any genetic resource from a foreign country particularly developing countries.
1.5.1 Bioprospecting
Bioprospecting is the search for economically valuable biochemical or genetic resources from living organisms for commercial or scientific purposes (Henne and Fakir,
1999). Typically this term is related to the search for resources with pharmaceutical or industrial potential but it can also be associated with the search for plants with ornamental horticulture potential. Whatever the primary purpose, bioprospecting is usually surrounded by controversy. From the perspective of the country or areas in which bioprospecting is taking place, these actions can have dual meaning. In some instances, bioprospecting is just a less threatening term for “biopiracy”, or the pillaging of the genetic resources of a country without fair compensation. Alternatively, ethical bioprospecting can be a positive entity as a potential source of income for developing countries that do not have the capability to develop their own genetic resources.
1.5.2 Convention on Biological Diversity
The opening of the Convention on Biological Diversity (CBD) in 1992 resulted from decades of individual initiatives to conserve particular species and ecosystems.
Developing countries desired a fair share of the benefits generated from any genetic resources originating from within their boundaries (Seiler and Dutfield, 2002). The CBD is the first multilateral agreement to address the issues of bioprospecting directly. The aims and goals of the convention are three fold: 1) the conservation of global
13 biodiversity; 2) sustainable use of the components of biodiversity; and 3) fair and equitable sharing of benefits arising from the use of genetic resources (Secretariat of the
Convention of Biological Diversity, 2001). The countries that join the convention are legally obliged to implement its provisions. Although the convention has established an international treaty to give the participating countries sovereign rights to determine how to regulate access to their national genetic resources, it is the responsibility of the individual member nations to provide critical leadership in establishing rules and guidelines.
1.5.3 Access and Benefit Sharing and Intellectual Property Rights
The CBD makes provisions that the acquired benefits resulting from use of conserved resources are to be “fairly and equitably shared.” It is important to note that benefits are not always just monetary shares such as bioprospecting fees or advance payments, but may also include joint publications, non-monetary benefits and services, training, and co-ownership of intellectual property rights (Seiler and Dutfield, 2002). To begin addressing the issues of access and benefit-sharing, the Third Conference of the
Parties for the Convention on Biological Diversity launched the Biotrade Initiative in
1996. This initiative engages a strategy that encourages mechanisms promoting and protecting intellectual property, traditional knowledge, and biodiversity, for national or local producers as well as local and indigenous communities (Rojas, 1999).
Access refers to resources as well as technologies and can be restricted by complicated legislation or by intellectual property rights (IPRs) (Seiler and Dutfield,
2002). Intellectual property rights are the rights to make, use, and sell a new product or
14 technology commonly patents, trademarks, or copyrights. Member countries of the CBD are expected to respect the protected IPRs that are part of an agreement. Seiler and
Dutfield (2002) explain, “A patent creates a legal monopoly that does not necessarily translate into a commercial one. But it definitely creates a barrier to access. In the CBD context, the real problem with these rights is therefore that the Convention’s technology transfer provisions- which are integral to benefit sharing- could effectively be undermined and even rendered meaningless”. Without the opportunity of technology transfer there remains little incentive for involvement of developing countries.
1.5.4 NBI- Ball Agreement: A working example
Prior to 1999, the existing bioprospecting agreements between companies of industrialized nations and developing countries had aimed at drug development for the pharmaceutical sector (Henne and Fakir, 1999). However, in August of 1999 Ball
Horticultural Company and the National Botanical Institute of South Africa signed a bioprospecting agreement that made it the first to aim only at the horticulture and floriculture sector (NBI, 2001; Henne and Fakir, 1999).
The NBI-Ball agreement set a new precedent for ethical bioprospecting in the filed of ornamental horticulture. It facilitated access to South Africa’s plants, in a manner that ensures fair and equitable sharing of benefits and transfer of technology (NBI, 2001).
NBI selects plants from its collections and presents them to Ball on the condition that
Ball pays royalties on the products that are developed from them and the plants are used for ornamental purposes only. The agreement established a benefit-sharing mechanism for the research and profit stage. During the research stage NBI is provided with an
15 initial research fee of $125,000 (US) and annual research fees starting at approximately
$28,000 (US) (Henne and Fakir, 1999). Ball in turn benefits with access to NBI researchers who maintain a knowledge and expertise of the native plant materials.
Benefits generated in the profit stage are generated from IPR protections. NBI retains exclusive right to use, sell or market plant materials within South Africa, however, Ball maintains the rights outside of South Africa but must exercise those rights in the name of the NBI (Henne and Fakir, 1999).
1.6 Production Research for a Type III Species
The potential for production research of type III species is vast considering that little is known about their growth control, flowering control and production practices.
Information pertinent to commercial production, such as propagation techniques, growth regulation, photoperiodic requirements, optimum temperatures, and insect interactions must also be fully explored and established before acceptance into the industry.
1.6.1 Propagation
Propagation is the controlled reproduction of plants. There are various methods of plant propagation, which can be divided into two basic categories, sexual and asexual.
The primary method of sexual propagation for floriculture crops is by seed with offspring bearing traits of both parent plants. Asexual propagation is plant reproduction by means of vegetative parts such as shoot, roots, or leaves. The genetic makeup of asexually propagated plants is identical to the parent plant. Asexual methods are used in lieu of seed propagation under three common circumstances: 1) offspring produced by seed are
16 too genetically variable for commercial production; 2) plants from seed are not produced in a reasonable amount of time; or 3) expensive and time consuming methods are required for seed germination and seedling development.
There is often little information concerning best means of propagation for type III species. Exploration into propagation methods is essential to determine if the species can be propagated and by what means (Armitage, 1986). If propagation by seed requires complex procedures to get a viable seedling or if there is only a small window of opportunity during which plants can only be successfully asexually propagated, further research in the development of the crop should be reconsidered.
Seed Dormancy
Dormancy is a condition that prevents the germination of a seed even when the environmental factors are suitable for germination. This condition is not uncommon and is often the largest barrier to domestication and cultivation of wild species (Knapp, 1990).
Seed dormancy is a survival adaptation for plants that results in the coordination of the timing a seed viability with the conditions most favorable for survival.
Primary dormancy includes exogenous and endogenous dormancy. Exogenous dormancy is non-embryo associated dormancy imposed by the enclosing tissues, such as endosperm, perisperm, or the seed coat. These tissues can inhibit water uptake, provide mechanical restraint to embryo expansion and radicle emergence, modify gas exchange, prevent leaching of germination inhibitors from the embryo, and supply inhibitors to the embryo (Bewley and Black, 1994). Perhaps the most common form of exogenous dormancy exists with seed with hard seed coats that become suberized and impenetrable
17 to water. Scarification, or mechanical abrasion of the seed coat, soaks in alcohol or other fat solvents to dissolve waxy substances that impede water entry, or soaks in concentrated acid (chemical abrasion) are three such methods to break dormancy in exogenous dormant species (Raven, Evert, Eichhorn, 1999). For a number of species in which the seed coverings are the primary barrier to germination, the embryo can be completely removed from the seed coat of the dormant seed and germinate normally.
Endogenous dormancy is associated with the embryo itself. Morphological endogenous seed dormancy occurs when embryos are not completely developed at the time they are shed from the maternal plant. Warm temperatures and treatment with gibberellic acid, a germination-stimulating hormone, is most often used to induce germination of morphological endogenous dormant seeds. Physiological endogenous dormancy is the most common form of seed dormancy (Baskin and Baskin, 1998). This type of dormancy involves physiological changes within the embryo that result in changes in its growth potential that allows the radicle to penetrate the seed coverings.
Physiological endogenous dormancy includes species that maintain a light or dark requirement for germination as well as those species whose seed must undergo a period of dry storage to loose dormancy.
Primary dormancy is a natural adaptation of a species to control the timing of germination using environmental cues. Secondary dormancy, however, is an adaptation to prevent germination of seed in unfavorable environmental conditions such as unfavorable temperatures, prolonged light or darkness, or water stress. Release from secondary dormancy can be induced by stratification (chilling), light, and in some cases treatment with gibberellic acid.
18 1.6.2 Control of Flowering
In order to ensure reproductive success, a species must maintain the capability to regulate flowering to coincide with optimal conditions for pollination and seed formation.
Developmental signals for flowering can include internal (or autonomous) factors, such as circadian rhythms and hormones or environmental factors, such as day length and temperature. A plant controlled by an autonomous system will flower strictly in response to internal developing factors regardless of any particular environmental condition.
Control of flowering by an environment-sensing system can be divided into two responses, obligate or facultative response. Obligate (or qualitative) response indicates that a plant has an absolute requirement for the proper environmental cues in order to flower. Flowering of a facultative (or quantitative) plant is promoted by a particular environmental condition but will eventually flower even in the absence of such cues. A facultative plant therefore relies on the functioning of both environmental and autonomous flowering systems. The two most important environmental conditions promoting a flowering response are photoperiodism and vernalization.
Photoperiodism
Photoperiodism, or plant flowering response to day length is an area of flowering research born in the early twentieth century through the work of USDA researchers W.W.
Garner and H.A. Allard (O’Neil 1992). Observations that “Maryland Mammoth” tobacco would not flower unless the day length was shorter than a critical number of hours led them to establish that the absolute duration of the light period controlled flowering response. Nearly a century later, research on photoperiod flowering response has grown
19 to encompass at least four areas: 1) physiological characterization of signal response; 2)
identification and role of phytochrome in photoperiodic timing mechanism; 3)
photoperiodic time measurement; and 4) identification of translocatable flower-
promoting and inhibiting substances (O’Neil 1992).
Classifying plants by their photoperiodic responses is usually based on flowering.
Distinguished by effects of day length on floral initiation, there are three general types of plants: (1) long-day plants (LDP) which flower only when day length exceeds a certain critical length (e.g., Arabidopsis, pea, Nicotiana sylvestris); (2) short-day plants (SDP)
which flower only when day length is less that the critical length (e.g., chrysanthemum,
soybean and N. tabacum cv. ‘Maryland Mammoth’); and (3) day-neutral plants (DNP)
which have no particular day length requirement for floral induction (e.g., tomato and N.
tabacum cv. ‘Wisconsin’). The term ‘critical day length’ does not refer to relative day
length but to whether the day length exposure is greater or less than that required by the
plant to flower. Absolute facultative day-length requirement is dependent on the
interactions between plant age, growth history, and environmental growth conditions to
control flowering.
Although the entire plant is sensitive, leaves most readily perceive light. Within
the leaf, light perception is initiated by at least three different photoreceptors:
phytochrome, which exists in two phytochromic forms, Pr and Pfr, absorbing red and far-
red light respectively; cryptochrome, a blue/UV-A absorbing pigment; and a UV-B
absorbing pigment (Vierstra, 1993; Furuya, 1993). Of these three photoreceptors,
phytochrome is the most prevalent and best characterized. Phytochrome detects the
relative amounts of red (R) and far-red (FR) in the plant growing environment and can
20 distinguish changes in the ratio of R to FR. Light quality, as it relates to plant growth is
often described in terms of the red to far-red light ratio. When leaves absorb red light,
phytochrome converts from the Pr to the Pfr form and converts back to Pr when FR is
absorbed (Thomas and Vince-Prue, 1997). The photoconversion between these two
phytochromic forms stimulates a morphological change allowing the plant to alter growth
and development in response to the light environment
Photoperiod is perceived by photoreceptor in the leaves, yet the morphological
changes associated with flowering occur at the apical meristem. Thus, it is assumed that
a floral response is triggered by biochemical signals originating in the leaves and
translocated to the apical meristem. To explain the phenomenon that photoperiodic
plants flower irrespective of the day length to which the apical meristem is exposed,
Chailakhyan (1937) proposed that the leaf-generated translocatable signal is hormonal in
nature called “florigen” (Bernier et al., 1985).
The basic evidence for the existence of this theorized floral stimulus is primarily
physiological. Early work with Perilla crispa showed that a single leaf held under
inductive (SD) photoperiod could be grafted in succession up to seven times resulting in
the induction of flowering of stocks held under non-inductive conditions (Zeevaart,
1969). Likewise, flowering of the day neutral N. tabacum cultivar ‘Trapezond’ held under short days was accelerated when grafted with a SD cultivar ‘Maryland Mammoth’
(Lang et al, 1977). Although neither of these studies was able to specifically identify a floral stimulating substance, both were a clear demonstration of the transmission of a floral promoting signal.
21 Vernalization
Plant flowering can also be controlled by temperature. Vernalization is the low
temperature promotion of flowering perceived at the vegetative shoot meristem. Plants
that require vernalization will remain vegetative without cold treatment. Flowering
response to cold temperatures typically requires several weeks of exposure, however, the
specific duration of low temperature is species dependent. The effective temperature
range for vernalization is from just below freezing to about 10ºC, with a broad optimum
usually between 1 and 7ºC (Lang, 1965). For many perennial plants, exposure to
vernalizing temperatures early in development significantly hastens flowering (Pearson et al., 1995). A study evaluating flowering of the half-hardy perennial Osteospermum
found that flower number increased when vernalized at temperatures 4-15ºC for 4 to 6
weeks (Suzuki and Metzger, 2001).
1.6.3 Growth Regulation
Plant growth regulation is defined as any chemical or process used to produce a
specific type of growth response, such as inhibition of internode elongation or root
development (Dole and Wilkins, 2005). These chemicals or process act to alter action of
endogenous plant hormones, auxins, gibberellins, cytokinins, ethylene, and abscisic acid.
Chemical Growth Regulation
Many greenhouse floriculture crops will undergo a triphasic pattern of plant
growth: 1) slow initial growth that occurs immediately after propagation; 2) rapid
vegetative growth and elongation; and 3) slow final reproductive growth during which
22 time flowers develop (Dole and Wilkins, 2005). For the effective use of chemical growth retardants, application must be before and during the rapid growth phase. Use of chemical growth regulators produces more compact plants with thicker stems, which allows the plant to better withstand handling and shipping.
Chemical plant growth retardants or plant growth regulators (PGRs) are synthetic compounds used to reduce the shoot length of plants in a desired way without altering development (Rademacher, 2000). These PGRs function by reducing the rate of cell division and cell elongation. Chemical growth regulators can be classified as either an ethylene releasing compound or inhibitors of gibberellin (GA) biosynthesis (Rademacher,
2000). Ancymidol, chormequat, daminozide, ethephon, and paclobutrazol are examples of the chemical growth retardants available for commercial use. Although all the chemicals used produce similar end results, the mechanism by which they alter the endogenous plant hormones is different. For example, paclobutrazol, daminozide, and chlormequat act as an inhibitors of gibberellin biosynthesis but are each inhibitors of different pre-cursors in the GA synthesis pathway. The effectiveness of any one PGR is species dependent due to varying levels of sensitivity.
Non-Chemical Height Control
With the rising concerns surrounding the negative impact of greenhouse chemical run-off on ground water sources and the environment, it is important that non-chemical means of growth regulation continue to be researched. The use of non-chemical methods of height control is commonplace for edible greenhouse crops, which have restrictions on
23 the use of chemical growth retardants. Commonly used non-chemical methods include alteration of light quality, container size restriction, nutrient restriction, water restriction, and temperature differential.
Both light quantity and light quality can affect plant height. Light, depending on crop requirement should be maintained at the highest level possible and plants must be spaced far enough apart to insure each plant will receive the maximum amount of light.
Altering the red (R) to far-red (FR) ratio of light can also have a significant impact on plant height. Light with a low R:FR (high in far red wavelengths) will cause plants to stretch and have longer internodes, while light with a high red to far red ratio (low in far red wavelengths) will encourage shorter plants with reduced internodes. A future production method to reduce the far-red wavelengths reaching the plant is the use of photoselective filters. Considerable work with herbaceous ornamental crops such as poinsettias (Clifford et al., 2004), chrysanthemums (Oyaert et al., 1999; McMahon,
1999), and potted miniature roses (McMahon and Kelly, 1990), have shown that growth under spectral filters reducing the transmission of far-red light results in reduced internode elongation and plant height.
Container size restriction, nutrient restriction, and water restriction are cultural practices that control plant height but with high associated risks if not properly monitored. Container restriction works on the premise that small media volume will restrict the growth by restriction of root growth (Dole and Wilkins, 2005). Although with this method precise control of irrigation application is required and there is the possibility of over stunting and quality reduction. Water restriction functions on the premise that systematic wilting but not allowing the plants to reach the wilting point can control the
24 height of some plant species. Nutrient restriction typically involves the restriction of
nitrogen levels particularly ammonium nitrogen, which promotes lush and rapid growth.
1.6.4 Temperature
The effect that the growing environment temperature has on plant growth and development can be a general and/or specific effect. A general effect of temperature would be the increase or decrease in plant growth rate as the temperature changes. An example of a specific temperature effect would be the induction of flowering by vernalization.
Each species has an optimum growing temperature range and a tolerable temperature range (Dole and Wilkins, 2005). The optimum temperature for a given species would be the temperature that produces the highest quality plant in the least amount of time. Tolerable temperatures permit the plant to grow but may reduce plant quality and/or increase production time. Plants held under temperatures falling in the lower tolerable temperature range will grow uneconomically slowly. Conversely, if the plants are held under high-end tolerable temperatures flower initiation can be delayed or quality is reduced.
Greenhouse temperature manipulation has been the only non-chemical method of growth regulation that has been widely applied in commercial production (Clifford et al.,
2004). There are two practices currently in use that employ altering the diurnal temperatures to control plant growth: 1) the difference (DIF) between day and night temperatures particularly when night temperatures are maintained higher than the day
25 temperatures; 2) a temperature drop for 2-4 hours after sunrise (Myster and Moe and references cited therein, 1995; Clifford et al., 2004).
DIF is the temperature differential obtained when the night (or dark period) temperatures are different from the day (or light period) temperatures. Plant growth response to the temperature differential is an example of a general response. The acronym DIF was coined by Dr. R. Heins of Michigan State University who uncovered the practical relationship between plant height and temperature differential (Nelson,
1998). DIF can be positive, negative, or zero. When the day temperatures are greater than night temperatures, DIF is a positive value. If the day temperatures are lower than the night temperatures, DIF is a negative value. DIF is zero when the day and night temperatures are equal. The relationship between these two temperatures has an effect on the elongation of the stem internodes and consequently the height of the plant. Plant height may be decreased by lowering the day temperatures or by raising the night temperatures. Conversely, raising the day temperature and lowering the night temperatures may increase plant height. This information is drawn from research conducted by Heins and Erwin (1990) controlling the height of Easter lily (Lilium longiforum) utilizing DIF. The Easter lilies were held under 26ºC day temperatures and were exposed to night temperatures of 14, 18, 22, 26, or 30ºC, giving DIF values of +12,
+8, +4, 0 and -4ºC, respectively. They found that as the DIF values increased, plant height also increased significantly. A significant number of other crops such as geraniums, impatiens, petunia, poinsettia, rose, snapdragon, sweet corn, tomato, and watermelon are very responsive to DIF as a mechanism of height control (Nelson, 1998).
26 1.6.5 Plant-Insect Interactions
In nature, plants and insects are just some of the living organisms that are continuously interacting in very complex ways (Mello and Silva-Filho, 2002). Such associations may be beneficial or detrimental to the plant. Plants provide insects with food, shelter, and a location to reproduce. The insects have several beneficial activities including defense and pollination. However, plant-insect associations can be detrimental to the host plant depending on the intensity of colonization and feeding.
When exploring the potential of a new floriculture crop it is important that close observation or studies be conducted to determine insect interactions with the species under study. With the growing interest of gardeners to attract birds and butterflies, determining the type of pollinators that a particular species would attract can provide an additional marketing tool. From the production prospective it is important to establish if a particular species exhibits high susceptibility or resistance to greenhouse pests such as aphids, thrips, mites, or whiteflies. High susceptibility to insect pests under production conditions could provide sufficient cause to consider removal of the new crop from a research program.
Plant Mechanisms of Insect Pollinator Attraction
A “symbiosis” exists between animal and insect pollinators and flowering plants.
Pollinators serve as the vectors for cross-pollination (and out-crossing) of plant species and are in turn provided with nutritive rewards. Such a relationship has promoted a co- evolution of flowers and their pollinators, which has resulted in the development of plant features that encourage visitation by particular pollinators. It is therefore no accident that
27 specific pollinators (animals or insects) are attracted to plants possessing particular flower structures, colors, or scents.
Butterflies, moths, and bees can be considered the most important of the insect pollinators. Butterflies and moths utilize both color and scent as attractants (Raven et al.,
1999). Butterfly pollinated species are brightly colored, specifically red and orange, and are sweetly fragrant. Because moths are nocturnal, flowering species that are pollinated by moths have a strong night fragrance and have a greater tendency to be white, which is the most visible color in darker conditions. Species often visited by moths and butterflies will tend to have a tubular structure with a long corolla tube allowing exclusive access to nectar by pollinators possessing the appropriate feeding organs.
Bees are incredibly important pollinators for many plant species and are dependent on color and distinguishing markings of flowers as guides to food. Plants utilizing bees as primary pollinators are brightly colored, usually blues and yellows (bees do not see red). Bees do have the ability to perceive ultraviolet light as a distinct color
(Raven et al., 1999). Therefore, some flowering plants have developed distinguishing coloring visible only within the ultraviolet range of the spectrum. Ultraviolet markings are an exclusive for bee pollinators. The smaller size of bee pollinators, with respect to others such as butterflies, moths or birds, plays a role in the fact that bees are less particular about the shape or structure of the flowers they visit.
28 Plant Mechanisms of Insect Herbivore Defense
To reduce the negative impact of attack, plants have developed different mechanisms of defense against herbivory. These different defenses include chemical and physical barriers that may be constitutive or induced mechanisms (Gatehouse, 2002).
A plant’s ability to manage attack by herbivores can be divided into two broad categories, resistance mechanisms and tolerance mechanisms. Resistance mechanisms are characteristics that reduce the damage inflicted upon plants by herbivores. Resistance mechanisms can be further categorized into deterrence or damage minimization factors.
Deterrence, or non-preference, factors refer to characteristics that lead insect herbivores away from a particular host by preventing plant recognition, colonization, or damage and can have either an allelochemical or morphological basis (Gatehouse, 2002). An allelochemic non-preference exists due to the presence of a chemical defense. Secondary metabolites have the chief function of protecting plants against herbivory. Plant secondary metabolites can be divided into three chemically distinct groups: terpenes, phenolics (tannins), and nitrogen-containing compounds (alkaloids) (Taiz and Zeiger,
1998). A morphological non-preference, on the other hand, results from plant structural characteristics that interfere with normal behavior of insects. Trichomes, as well as spines and thorns are examples of such physical defenses. Although trichomes also function to lower leaf temperature, decrease water loss, and increase water absorption they also provide a significant line of defense against insects. Depending on the morphology of the trichomes, defense may be due to a barrier to food source imposed by a high density of trichomes, physically wounding the insects, or secreting a deterring or toxic chemical resin (Kelsey et al., 1984).
29 Damage minimization, or antibiosis, is the second type of resistance mechanism factor. Antibiosis refers to all adverse effects on the insect, which result in minimized losses for the plant. Some of the effects of antibiosis on insect herbivores are: reduced survival, reduced growth rate, reduced size, shortened adult life span, reduced fecundity, morphological abnormalities of offspring.
The second broad category of plant defense mechanisms is tolerance. Tolerance mechanisms are responses resulting in the ability of the plant to withstand insect infestation and reduce the negative impact of injury. Unlike resistance mechanisms, only plant response is involved in tolerance. Tolerance mechanisms fall into two subcategories: physiological tolerance mechanisms and morphological tolerance mechanisms. Physiological tolerance mechanisms are alterations of the plants physiology in response to injury to compensate for the damage. Such mechanisms might involve capability to “outgrow” the damage or involve the storage of carbohydrate and nutrients to draw from in the event of tissue loss. Morphological tolerance mechanisms can include maintenance and protection of multiple meristems to compensate for lost tissue and ability to follow an alternative developmental pathway depending on the intensity of herbivory.
30 1.7 Research Objectives
No information exists regarding the horticultural qualities of Cuphea pulchra and C. schumannii. The focus of the present research was to evaluate the two species as landscape or garden ornamental plants and begin development of information pertinent to commercial production. The general objectives of the research were:
1. Evaluate performance of C. pulchra and C. schumannii as annual ornamentals;
2. Determine the most economically feasible means of propagation;
3. Determine temperature effects on growth and development and begin defining the
optimal temperature range for production;
4. Evaluate chemical and non-chemical methods of growth regulation;
5. Determine photoperiodic requirements for flowering; and
6. Evaluate C. pulchra mechanism of resistance to greenhouse pests and investigate
the role of glandular trichome exudate compounds in the resistance.
31 CHAPTER 2
INITIAL OBSERVATIONS OF PERFORMANCE UNDER GREENHOUSE
AND SIMULATED LANDSCAPE CONDITIONS
2.1 Introduction
For many new floriculture crops, particularly those that are a nondomesticated genera or species, very little or no cultural information exists. Information regarding propagation methods and growth and developmental response under greenhouse or outdoor environmental conditions must be determined. The challenging task of a new crop researcher is beginning with a blank slate and developing the evaluations necessary to accumulate pertinent production information.
Once plant material has been acquired, it takes approximately one year, to build up enough stock plants to begin experimentation. Roh and Lawson (1988) label this period as the initial observation period. During this time propagation methods are explored as well as monitoring plant appearance, growth habit, flowering, and susceptibility to pest and diseases under production conditions.
When developing and evaluating a species for introduction as a potential landscape or garden plant, information regarding species response to an outdoor growing environment is necessary. Outdoor conditions can be more harsh and variable in comparison to greenhouse conditions, which may result in differences in plant growth
32 and development. Therefore, outdoor trialing is important to evaluate species response under outdoor environmental conditions. Trials also provide the opportunity to collect data pertinent labeling information for a garden plant such as maximum expected plant height, flowering time, and cultural requirements.
The susceptibility to physiological disorders is a very important characteristic for which plants much be evaluated during an initial period of observation. High susceptibility to a physiological disorder, pest, or disease would give just cause for removal of the species from a research program. Development of leaf intumescence, or oedema, is a physiological disorder that is prevalent for many species grown under controlled environmental conditions. Intumescence injury is characterized by galls, 1-
3mm in diameter between major veins of the leaves (Lang et al., 1983). They are caused by non-pathogenic factors only in controlled environments such as greenhouses and growth chambers. Intumescence injury has been found to affect tomato (Lang and
Tibbitts, 1983), potato (Seabrook and Douglass, 1998), and geranium (Metwally et al.,
1970). Jaworski et al. (1988) evaluated sixteen species of Cuphea for development of leaf intumescence under greenhouse conditions. For six of the species, the percentage of leaves exhibiting intumescence injury ranged from 20% to 100%.
The current research sets out to evaluate two wild species of the genus Cuphea, C. schumannii and C. pulchra as potential new crops for the ornamental horticulture industry. No information exists regarding the horticultural qualities of either of these species. To determine what experimentation and in-depth evaluation may be required to prepare these species for introduction, a period of initial observation was necessary. The
33 initial observation period paralleled the average one-year span as approximated by Roh
and Lawson (1988) from autumn of 2000 through autumn 2001.
The objectives of this period of initial observations were to: 1) determine what
type of plant culture these Cuphea species would be best suited for (e.g. pot culture or
bedding plants); 2) determine the best means of propagation (discussed in Chapter 3); 3)
observe flowering response under greenhouse and outdoor environmental conditions; and
4) observe species susceptibility to pests, disease, and/or physiological disorders. These
objectives were accomplished through observation of stock and propagated plant material
in the greenhouse, as well as establishing trails to evaluate species performance as
bedding plants under outdoor environmental conditions.
2.2 Materials and Methods
2.2.1 Initial Greenhouse Observations
Plant Culture
Stock plants of C. schumannii were started in 1999 from seed provided by Dr.
Shirley A. Graham, professor at Kent State University. The seed was pooled from C.
schumannii plants maintained in the Kent State University biological greenhouses from
1991-1999. The parent plants were from seed collected in Oaxaca, Mexico. Stock plants
of C. pulchra were started in 1999 from seed collected in January 1998 in Bahia, Brazil by Dr. Shirley A. Graham.
Cuphea stock plants were maintained in The Ohio State University Department of
Horticulture and Crop Sciences greenhouse. Plants were held under a 16-hour
34 photoperiod (600-2200 HR) using supplemental lighting from 1000 W metal halide
lamps (Hydrofarm, Petaluma, CA). When the natural light intensity fell below 200
W m-2, the supplemental lighting provided a photosynthetic photon flux (PPF) of 100
µmol m-2. Daily watering was facilitated by an automatic drip tube irrigation system.
Plants were fertilized twice a week with 200 mg N L-1 of 20-10-20 Peters complete
fertilizer (Scotts Co., Marysville, OH). Plants were treated with insecticides and
preventative fungicides as needed.
Stock plants were vegetatively propagated for use in various experiments. Growth
and development of plants under standard greenhouse conditions were observed.
Observations included plant growth habit in containers, flower and leaf morphology and
pest susceptibility.
Media Moisture Effects on C. schumannii Leaf Intumescence
Trial 1. The first trial to evaluate media moisture effects of leaf intumescence was initiated on May 12, 2003. One week prior to initiation, 20 C. schumannii cuttings were transplanted into 15.24 cm diameter standard plastic pots and transferred to a Conviron growth chamber (Winnipeg, Canada). Growth chamber conditions were 16 hours of approximately 260 µmol m-2s-1 light provided by incandescent and fluorescent bulbs with
a 21ºC light 18ºC dark cycle.
The trial was conducted under glass in the OSU Horticulture and Crop Science
greenhouse. Plants were divided into four watering treatment groups: 1) pots set in a
saucer to hold leaching water and watered every day, 2) pots watered every day (no
saucer), 3) pots watered every two days (no saucer), and 4) pots watered every three days
35 (no saucer). The starting height, number of expanded leaves, and number of leaves with
intumescence were recorded for all plants. At the initiation of the experiment, all pots
were watered to saturation (water leaching out the bottom of the pot). On the designated
day of watering, each replicate in the treatment was weighed. Plants were watered to
saturation, allowed to drain for 15 minutes, and then weighed again. After three weeks,
final plant height, number of expanded leaves, number of expanded leaves with
intumescence, leaf area using a Li-Cor 3100 leaf area meter (Lincoln, Nebraska), and
plant dry weight were recorded. A one-way ANOVA was used to analyze treatment effects on each of these parameters using Sigma Stat Statistical software (Jandel
Corp./SPSS, Chicago, IL).
Trial 2. The second trial to elucidate media moisture effects of leaf intumescence was initiated on July 25, 2003. All methods were the same as those described for Trial 1 with the exception that the water treatment groups were: watered every day, watered every three days, and watered every five days. A one-way ANOVA was used to analyze treatment effects on measured parameters.
2.2.2 Outdoor Trials
2001 Outdoor Simulated Landscape Trial
C. schumannii and C. pulchra plants were started from vegetative cuttings taken from stock plants and transplanted into 20 cm diameter squat pots approximately three months before initiation of the outdoor trial. Two weeks prior to planting, C. schumannii and C. pulchra were moved from the greenhouse to an outdoor area covered by woven shade cloth to acclimate. On May 31, 2001, the Cuphea were planted in the raised 3.05
36 m x 6.1m trial bed containing commercial topsoil. The bed had a north-south orientation providing full sun exposure. A planting design was incorporated into the trial allowing for 61cm plant center spacing. Plants in the trial were watered as needed by overhead irrigation and fertilized approximately every three weeks with 200 mg N L-1 of 20-10-20
Peter’s complete fertilizer (Scott’s Co., Marysville, OH) through late September.
Maintenance such as weeding, moderate pruning and flower deadheading was performed on an as needed basis. Climate data for the trial period was collected using a Campbell Scientific ET 106 weather station (Logan, UT) located approximately
15 m from the trial bed (Table 2.1).
Maximum Minimum Total Temperature Temperature Precipitation Month (ºC) (ºC) (mm) June 29.4 16.6 52.6 July 30.4 18.6 81.3 August 30.1 18.2 122.4 September 24.8 11.0 45.5 October 19.0 7.1 70.6 November 15.7 3.3 83.1
Table 2.1. 2001 trial climate data. Average monthly maximum and minimum air temperature and total rainfall for June through November 2001. Data collected by a Campbell Scientific ET 106 weather station.
37 In mid October, a 6.4 cm layer of hardwood mulch was laid on the bed and plants were moderately pruned in preparation of over-wintering the plants. After this point, no further maintenance was performed.
Plant evaluations were conducted every three weeks beginning three weeks after planting through early October. Digital pictures were taken to document trial progress and changes in individual plant appearance and growth. Plants were also observed for pest susceptibility and presence of active pollinators.
2002 Outdoor Simulated Landscape Trial
C. schumannii and C. pulchra plants were started from vegetative cuttings taken from stock plants on March 6, 2002. At four weeks, cuttings were transplanted into
15.24 cm diameter standard pots and maintained under greenhouse conditions. Two weeks prior to planting, C. schumannii and C. pulchra were moved from the greenhouse to an outdoor area covered by woven shade cloth to acclimate. On May 15, 2002, plants of both species were planted in the same raised bed as the 2001 trial. Planting was done in rows giving each plant a 61 cm planting center. Plant maintenance was similar to that in 2001; however, Miracle-Gro 30-10-10 fertilizer for acid loving plants (Scotts Co.,
Marysville, OH) was applied approximately every three weeks using 7.6 liter watering cans at a rate of 1 tablespoon of soluble fertilizer per 3.8 liter.
Prior to planting, five soil core samples were collected from the raised planting bed and analyzed to determine soil pH. An average pH level above 7 prompted the application of aluminum sulfate granules to acidify the soil. Most flowering crops grow
38 best in a slightly acid pH range of 6.2 to 6.8 in soil-based substrates (Nelson, 1998). An application rate of 2.27 kg aluminum sulfate per 30.48 m2 was necessary to drop the soil pH one full unit.
Plant height and plant width in perpendicular directions (to calculate plant diameter) were recorded for each plant on June 7, June 28, July 19, August 20,
September 11, and October 2. Digital images were taken to document trial progress and changes in individual plant appearance and growth on May 15, June 19, July 3, 17, 31,
August 21, September 4, 19, October 3, 23, and November 6. Climate data for the trial period was collected (Table 2.2).
Average Maximum Minimum Total Temperature Temperature Temperature Precipitation Month (ºC) ( ºC) ( ºC) (mm) May 15.4 22.1 9.2 139.2 June 23.8 30.3 17.7 89.7 July 25.9 32.4 19.8 66.6 August 24.8 31.4 18.9 39.4 September 21.8 29.1 15.2 117.1 October 12.0 17.3 7.2 67.8 November 5.4 9.7 1.6 72.4
Table 2.2. 2002 trial climate data. Average, maximum, and minimum air temperature and total rainfall for May through November 2002. Data collected by a Campbell Scientific ET 106 weather station.
39 2003 Outdoor Simulated Landscape Trial
C. schumannii and C. pulchra cuttings taken from stock plants on March 7, 2003 underwent the same pre-planting, planting and maintenance regime as those in 2002.
Cuphea were planted in the raised bed on May 27, 2003.
The planting bed was prepared by removing the layer of mulch applied in the fall of 2001. Soil pH testing indicated that pH levels were below 6.5, therefore, no aluminum sulfate amendment was necessary. Organic material into was added to the planting bed media. An approximately 7.62 cm thick layer of a 60% pine, 25% peat, 7% sludge, 7% haydite, 1% sand media mix (Willoway Nurseries Inc., Avon, OH) was applied to the entire bed and tilled into the existing top soil.
Plant height and plant width in perpendicular directions (to calculate plant diameter) were recorded for each plant on May 28, June 18, July 9, 29, August 27,
September 17, and October 8. Digital pictures were taken to document trial progress and changes in individual plant appearance and growth on those same dates. Climate data for the trial period was collected (Table 2.3). Cumulative photosynthetic photon flux (PFF) was monitored (Table 2.4) in the raised bed trail area using a quantum light sensor and data logger (Spectrum Technologies, Inc., Plainfield, IL).
40 Average Maximum Minimum Temperature Temperature Temperature Total Rain Fall (ºC) (ºC) (ºC) (mm) May 16.6 22.3 11.5 160.3 June 20.5 26.5 14.6 55.1 July 23.3 29.5 17.9 99.6 August 23.5 30.0 18.4 224.0 September 18.5 24.9 12.7 150.4 October 12.1 18.8 6.0 41.1 November 8.8 14.0 4.0 66.3
Table 2.3. 2003 trial climate data. Average, maximum, and minimum air temperature and total rainfall for May through November 2003. Data collected by a Campbell Scientific ET 106 weather station.
Monlthy Cumulative -2 -1 Month PFF (mol·m ·s ) June 0.23 July 0.33 August 0.27 September 0.22 October 0.16
Table 2.4. Monthly cumulative photosynthetic photon flux exposure of C. pulchra and C. schumannii trial plants under full sun conditions for June through October 2003.
41
2.3 Results and Discussion
2.3.1 Initial Greenhouse Observations
Potential as Potted Crops
Potted plant crops are those that are grown and produced for indoor (and
sometimes outdoor container) use. Common examples of potted plant crops are
poinsettias, Easter lily, orchids, and florist mums. Neither C. pulchra nor C. schumannii possess potential as a potted flowering crop. There are quite a few characteristics that make these two species unsuitable for pot culture. C. schumannii, in particular has a
“leggy” growth habit that is not well contained in pot culture (Figure 2.1). Although C.
pulchra maintains a more compact growth habit that might be suitable for pot culture, the
glandular trichomes of Cuphea pulchra make it a very sticky plant not be well suited for indoor conditions. The flowers of both species produce copious amounts of nectar that are stored in the spur at the based of the floral tube. When floral tubes are bumped or the plant is jostled, this sticky nectar will drip out of the floral tube onto clothing or nearby surfaces.
Although the ultimate horticulture use for C. schumannii and C. pulchra will not be as potted flowering crops, cuttings and transplants perform well in 15.2 cm diameter standard pots for production as potted bedding plants.
42
Figure 2.1. Cuphea schumannii maintains a “leggy” and indeterminate growth habit making it unsuitable for production as a potted crop. This characteristic along with susceptibility to greenhouses pest and physiological disorder make C. schumannii a poor candidate for introduction into the floriculture industry.
43 Cuphea pulchra Lines
Three stock plants of C. pulchra were grown out from seed collected directly from native plants in Bahia, Brazil. As the plants matured, differences in growth habit and morphology became obvious. All lines can be considered suffrutescent with a woody base and herbaceous branching, however growth habit and leaf morphology differ. These stock lines were given the letters “A”, “B”, and “C” to distinguish among them. C. pulchra “A” maintains a rounded and more compact growth habit with upright,
“soft” branching giving the plant a delicate appearance (Figure 2.2A, B). C. pulchra “B” growth habit is variable with a spreading habit that is a more free branching form than line “A”. The herbaceous branches and leaves are stiff and slightly thicker (Figure 2.3A,
B). C. pulchra “C” maintains a free branching, sprawling growth habit, leaves are stiff, similar to line “B” yet have slightly scalloped margins (Figure 2.4A, B). C. pulchra
“white” is not a line of C. pulchra but should be considered a mutation (or sport) of C. pulchra. “A”. It arose as a mutated stem bearing deep green leaves and white-pale pink flowers from the C. pulchra “A” stock plant (Figure 2.5A, B) C. pulchra “white” has been vegetatively propagated through four generations and continues to maintain the same characteristics. With the exception of floral tube color of C. pulchra “white”, floral morphology does not appear to be variable between lines (Figure 2.6A, B). Outdoor trials confirmed that these lines hold the same growth habit and morphological characteristics under outdoor environmental conditions.
44
Figure 2.2. A. Cuphea pulchra line “A” maintains a rounded, compact, upright growth habit. B. Stems and leaves of C. pulchra “A” are soft and flexible which give the plant a delicate appearance.
Figure 2.3. A. Cuphea pulchra line “B” maintains a spreading yet more open and free form than C. pulchra “A”. B. Stems and branches are thick and leaves are stiff, giving the plant a rigid appearance.
45
Figure 2.4. A. Cuphea pulchra line “C” maintains a free branching, sprawling growth habit. B. Branching and leaves are stiff similar to line “B”, however, leaves have finely scalloped margins.
Figure 2.5. A. Cuphea pulchra “white” is a sport of line “A” maintaining the same rounded, compact, and delicate growth habit. B. Foliage of “white” is a deeper green than its parent plant and the floral tubes are whitish to light pink color.
46
Nectary or “disc” Capsule (contains placenta)
Spur
Pedicle A
Glandular Trichomes
Floral ribs (vasculature)
Emerging Stigma
Stamen
B
Figure 2.6. A. Lateral cutaway view of inner floral tube of Cuphea pulchra. B. Lateral view of the mouth of the floral tube of C. pulchra.
47 Pest resistance
Within the first few months of observation, it became apparent that difference
existed between the two species with respect to greenhouse pest resistance. C.
schumannii plants were very susceptible to green peach aphid (Myzus persicae) (Figure
2.7) and western flower thrips (Frankliniella occidentalis) infestation,
Figure 2.7. Cuphea schumannii susceptibility to green peach aphid (Myzus persicae) infestation under greenhouse conditions. This image of an approximately 4cm2 area of the underside of a fully expanded C. schumannii leaf shows seven adult and approximately forty-three visible nymph green peach aphids illustrating the species high susceptibility to infestation.
particularly on new growth and flowers. Thrips control was particularly difficult because
of their ability to hide in the tubular flowers. However, C. pulchra plants maintained in
the same greenhouse room exhibited no signs of susceptibility to either aphid or thrips.
48 With respect to visible defense mechanisms, the significant difference between the two species is the presence of glandular secreting trichomes. C. schumannii lacks glandular trichomes on all plant surfaces. C. pulchra however maintains a dense concentration of glandular trichomes on stems and flowers. This observation led to the design of experiments to confirm the resistance of as well as elucidate the role of glandular trichomes of C. pulchra (discussed in Chapter 7).
Cuphea schumannii Leaf Intumescence
Leaf intumescence or oedema is a physiological disorder that develops almost exclusively in enclosed environments such as greenhouses or growth chambers (Morrow and Tibbitts, 1987). During initial greenhouse observations, C. schumannii exhibited significant intumescence injury on leaves. The predominant explanation for this injury under these types of conditions is that it occurs under conditions of high relative humidity, high soil moisture, and cool weather, which lead to an excess of water in leaf tissues (Metwally et al, 1971). As a small experiment in June of 2002, three C. schumannii plants that had been outside were moved into the greenhouse. Within four days, each plant was exhibiting intumescence injury (Figure 2.8).
Studies evaluating the development of intumescence in geranium identified excessive soil moisture as a main cause of leaf oedema formation in the species
(Metwally et al., 1970). Therefore, an experiment was designed to evaluate soil moisture as a causal factor in the development of intumescence on C. schumannii foliage. The results of the first trial indicated no significant treatment effects on final plant height,
49
50
Figure 2.8. Development of intumescence injury on the foliage of Cuphea schumannii under controlled environmental conditions of the greenhouse. The image on the left is a C. schumannii plant that had been growing outdoors in a 15.24cm diameter pot. There is no evidence of intumescence injury on the leaves. The image on the right is the same plant four days after being transferred into the greenhouse. Intumescence injury develops along and between the veins in the leaves and leaves begins to curl under and distort.
number of expanded leaves, number of expanded leaves with intumescence, flower number, leaf area, or plant dry weight for plants watered everyday, plants watered every two days or plants watered every three days (Table 2.5). The second trial did produce some significant treatment effects for some of the plant parameters measured (Table 2.6).
However, statistical significance of plants watered every three days is due in large part to the unexplainable difference in plant stature particularly height and average number of expanded leaves. It is not possible to conclude with certainty that soil moisture played a significant role in these differences. There was a noticeable difference in the percentage of expanded leaves displaying intumescence injury. One hundred percent of leaves in plants watered every day exhibited intumescence injury, whereas plants watered every three days and every five days averaged 88.8% and 88.9% intumescence injury, respectively.
Based on the results of the two trials, it is difficult to conclude with certainty that excessive soil moisture is the major cause of intumescence injury of C. schumannii. This type of injury is most likely due to a combination of factors working simultaneously.
Under greenhouse conditions, it is very difficult to tightly control these factors, particularly relative humidity. Therefore, it will be necessary in future research to design experiments in which environmental variables light, relative humidity, temperature, and soil moisture must be held constant while manipulating only one variable at a time.
51
# of Fully % Ful l y Expanded Change in Height Expanded Leaves Leaves with 2 Watering Treatment (cm) per Plant Intumescence Leaf Area (cm ) Plant Dry Wt. (g) Everyday (with saucer) 25.9 ± 1.5 24.8 ± 2.9 95.9 ± 2.0 647.9 ± 40.3 6.2 ± 0.5 Everyday 26.5 ± 0.9 24.4 ± 0.9 95.8 ± 1.9 519.3 ± 24.4 5.1 ± 0.3 2 Days 25.2 ± 2.2 21.0 ± 1.7 93.4 ± 3.1 564.1 ±49.5 5.0 ± 0.5 3 Days 24.5 ± 1.6 21.6 ± 1.6 96.0 ± 3.0 607.2 ± 50.1 4.9 ± 0.8 No significant treatment effects (P<0.05) 52
Table 2.5. Effects of soil moisture on development of leaf intumescence and other growth parameters of Cuphea schumannii (Trial 1). C. schumannii treatment groups were watered everyday with and without a saucer to catch draining water, every two days (no saucer), or every three days (no saucer). Values are treatment means ± standard error. Change in height was calculated by subtracting the initial height from final height. No significant difference existed between the treatment groups for any parameter.
# of Fully % Expanded Leaf Area of Change in Height Expanded Leaves Leaves with Expanded Leaves Watering Treatment (cm) per Plant Intumescence (cm 2) Plant Dry Wt. (g) a a a a a b Everyday 22.7 ± 1.7 15.2 ± 1.4 100.0 ± 0.0 412.3 ± 17.3 2.9 ± 0.1 3 Days 9.1 ± 3.1 b 8.2 ±1.2 b 88.9 ± 4.4 a 207.6 ± 65.4 a 1.6 ± 0.3 a 5 Days 22.2 ± 4.9 a 12.5 ± 1.8 a b 88.9 ± 5.1 a 410.0 ± 80.1 a 3.1 ± 0.6 b Values followeda,b by the same letter are not significantly different at P<0.05
53 Table 2.6. Effects of soil moisture on development of leaf intumescence and other growth parameters of Cuphea schumannii (Trial 2). C. schumannii treatment groups were watered everyday, every three days, or every five days. Values are treatment means ± standard error. Change in height was calculated by subtracting the initial height from the final height. The different watering treatments produced no significant difference in the percent of leaves exhibiting intumescence injury suggesting that soil moisture may work in conjunction with other environmental factors such as relative humidity and temperature to induce intumescence injury under controlled conditions.
2.3.2 Outdoor Trials
2001 Trial Observations
The landscaping value of a bedding plant is determined by its foliage, bloom color, duration of bloom time and resistance to pests and disease. Despite the allowance of the approximate 61 cm center spacing in the planting design, no accurate growth measurements could be collected for the greater part of the growing season due to plants growing into one another. Therefore, this first season failed to provide usable data in regard to optimum plant spacing and expected plant size. Nevertheless it was clear that both C. schumannii and C. pulchra responded very well to the growing environment. All specimens of both species exhibited vigorous growth throughout the season with a particularly significant increase during the month of July and beginning to slow in early
September (data not available). All plants continued to bloom through the growing season. Some of the C. pulchra specimens were able to maintain a significant amount of blooms and color as the plant focus into as late as mid November. As C. schumannii continued to mature the focal point was more on the large foliage than the inflorescence.
Early in the season, it was noted that the new growth of some of the trial plants, particularly C. pulchra, was exhibiting chlorosis. Soil analysis indicated that the soil pH level was about 7.8. This confirmed first assumptions that chlorosis might be the result of the plant inability to take up micronutrients, particularly iron, due to high soil pH.
Symptoms of chlorosis were alleviated by aluminum sulfate application (2.27 kg/9.29 m2) to the soil surface, which lowered soil pH approximately 1 unit. Plants were also fertilized with Miracid® at a rate of 1 tablespoon soluble fertilizer per 3.8 L. Within two weeks, symptoms of chlorosis declined.
54 Both species possess the pollinator attracting features of brightly colored floral tubes, and in the case of C. schumannii, brightly colored petals. These species also reward their visitors with a copious amount of nectar. Frequent pollinators of the plants were bumblebees, honeybees and yellow jackets (Figure 2.9A, B) Humming birds were frequently observed visiting the C. pulchra in two home garden locations (Leopold et al.,
2003). However, C. schumannii became the targets of nectar robbing. Bees were able to make slits in the rear, dorsal portion of the floral tube to gain direct access to nectar reserves in the spur (see Figure 2.6A). The drawback to nectar robbing is tissue necrosis around the slits made by the bees in the floral tube results in unsightly flowers (Figure
2.10). A probable reason why C. pulchra is not subject to nectar robbing is due the high density of sticky glandular trichomes on the exterior floral tube (see figure 2.6B), which deter bees from settling on the flower surface.
AB
Figure 2.9. A. Bumblebee on Cuphea pulchra flower. B. Honeybee and yellow jacket wasp on Cuphea schumannii flower. All insect pollinators were observed visiting both species. 55
Figure 2.10. Nectar robbing from flowers of C. schumannii. Bees were capable of making an incision through rear dorsal portion of the floral tube (indicated by arrow) to gain direct access to the nectaries. Necrosis of the floral tissue at the cite of the incision creates unattractive flowers
It is also worthy to note that minimal damage by outdoor insect pests was
observed on either species. No insect damage was noted on C. pulchra throughout the
season. This is not unexpected when considering that under greenhouse conditions, C.
pulchra remained free from greenhouse pests. Despite susceptibility to greenhouse pests,
C. schumannii in the trial exhibited only minimal foliar damage only by Japanese beetles.
No other insect pest injury was observed. Ants were also profuse on the C. schumannii
because of easy access to flower nectar but did not cause any visible damage to the plant.
Both C. schumannii and C. pulchra are perennial plants in their native habitat.
However, no plants of either species over-wintered in Zone 5. Plants were pulled in
April 2002. Inspection of the roots confirmed that neither C. schumannii nor C. pulchra
56 were able to maintain viable root for spring regeneration. However, by early June 2002,
a significant number of seedlings of both species began emerging in the bed, indicating
that some seed from the 2001 trial remained viable through the winter.
2002 Trial Observations
Planting Cuphea in rows permitted collection of usable data concerning optimum plant spacing and expected plant size during the 2002 trial season. C. pulchra lines “A”,
“B”, and “C” all exhibited increasing height (Figure 2.11) and diameter (Figure 2.12)
through the trial season. For lines “A” and “B”, height increase rapidly until mid July
whereas line “C” continued rapid increase until mid August. By mid September all lines
exhibited nearly the same height. Plant diameter of C. pulchra lines “B” and “C”
increased steadily until mid September, at which point they began to plateau for the
season. C. pulchra “A”, however, exhibited only a comparatively small increase (less
than 30 cm) in diameter over the growing season. Because of the rounded and compact
nature of this line, this is an expected observation.
Figure 2.13 and Figure 2.14 compare the height and diameter change,
respectively, of C. schumannii started by seed and by cutting during the 2002 outdoor
trial. Because of the “leggy” nature of C. schumannii, the plant diameter measurements
can not an accurate indicator of the plant shape. Cuttings of C. schumannii consistently
maintained increased height over those started from seed. The significant decrease in
average diameter and height in the C. schumannii between the July and August data
collection date can be explained by a wind and rain storm that caused mechanical damage
57 and stem breakage. Plants started by seed seemed to have been only mildly affected, owing in part to their smaller stature. All other observations made with regard to flowering, pests, and pollinators paralleled observations from the 2001 outdoor trial.
45
40
35
30
25
Height (cm) 20 C. pulchra B C. pulchra A 15 C. pulchra C
10 6/7/2002 6/28/2002 7/19/2002 8/20/2002 9/11/2002 10/2/2002
Figure 2.11. Cuphea pulchra lines “A”, “B”, and “C” height data collected during the 2002 summer trial from June 7, 2002 through October 2, 2002. Each point represents the mean height of four plants of each line. Vertical bars indicate standard errors of the mean.
58 100
90
80 . 70
60
50
40 Diameter (cm)
30 C. pulchra B C. pulchra A 20 C. pulchra C 10 6/7/2002 6/28/2002 7/19/2002 8/20/2002 9/11/2002 10/2/2002
Figure 2.12. Cuphea pulchra lines “A”, “B”, and “C” plant diameter data collected during the 2002 summer trial from June 7, 2002 through October 2, 2002. Each point represents the mean diameter of four plants of each line. Vertical bars indicate standard errors of the mean.
55
50
45
40
35
30 Height (cm) Height 25
20 Cutting Seed 15
10 6/7/2002 6/28/2002 7/19/2002 8/20/2002 9/11/2002 10/2/2002
Figure 2.13. Cuphea schumannii plant height data collected during the 2002 summer trial from June 7, 2002 through October 2, 2002. Each point represents the mean height of eight plant started by cutting and three plants started from seed. Vertical bars indicate standard errors of the mean.
59 70
60
50
40 Diamter (cm) 30 Cutting 20 Seed
10 6/7/2002 6/28/2002 7/19/2002 8/20/2002 9/11/2002 10/2/2002
Figure 2.14. Cuphea schumannii plant diameter data collected during the 2002 summer trial from June 7, 2002 through October 2, 2002. Each point represents the mean diameter of eight plant started by cutting and three plants started from seed. Vertical bars indicate standard errors of the mean.
2003 Trial Observations
Observations of C. pulchra grown in 2003 closely mirrored what was observed
during the 2002 trial of increasing height (Figure 2.15) and diameter (Figure 2.16) of lines throughout the trial season. The 2003 trial also included C. pulchra “white”
specimen. C. pulchra “white” maintained a growth pattern parallel, though slightly less
than its parent C. pulchra “A”.
Only C. schumannii cuttings were included in the 2003 trial. Both height (Figure
2.17) and diameter (Figure 2.18) of C. schumannii increased steadily through the trial
season and began to taper off between the middle of September and early October.
60 50
45 40
35
30
25
Height (cm) 20 C. pulchra A 15 C. pulchra white C. pulchra B 10 C. pulchra C 5
0 5/28/2003 6/18/2003 7/9/2003 7/29/2003 8/26/2003 9/17/2003 10/8/2003
Figure 2.15. Cuphea pulchra lines “A”, “B”, “C”, and “white” height data collected during the 2003 summer trial from May 28, 2003 through October 8, 2003. Each point represents the mean height of three plants of each line. Vertical bars indicate standard errors of the mean.
90
80
70
60 )
50
40 Diameter (cm Diameter 30 C. pulchra A C. pulchra white 20 C. pulchra B 10 C. pulchra C
0 5/28/2003 6/18/2003 7/9/2003 7/29/2003 8/26/2003 9/17/2003 10/8/2003
Figure 2.16. Cuphea pulchra lines “A”, “B”, “C” and “white” plant diameter data collected during the 2003 summer trial from May 28, 2003 through October 8, 2003. Each point represents the mean diameter of three plants of each line. Vertical bars indicate standard errors of the mean.
61 60
50
40
30 Height (cm) Height 20
10
0 5/28/2003 6/18/2003 7/9/2003 7/29/2003 8/26/2003 9/17/2003 10/8/2003
Figure 2.17. Cuphea schumannii plant height data collected during the 2003 summer trial from May 28, 2003 through October 8, 2003. Each point represents the mean height of nine plants started by cutting . Vertical bars indicate standard errors of the mean.
120
100
) 80
60 Diameter (cm Diameter 40
20
0 5/28/2003 6/18/2003 7/9/2003 7/29/2003 8/26/2003 9/17/2003 10/8/2003
Figure 2.18. Cuphea schumannii plant diameter data collected during the 2003 summer trail from May 28, 2003 through October 8, 2003. Each point represents the mean diameter of nine plants started by cutting . Vertical bars indicate standard errors of the mean. 62 2.4 Conclusions
Based on early greenhouse observations and outdoor trial observations, it
becomes clear that Cuphea pulchra possesses significant potential as a new floriculture
crop. Initial observations of performance of C. pulchra under greenhouse conditions
indicated the need for further evaluation of photoperiodic requirements, temperature
requirements for growth and flowering, growth regulation during production, and the
basis of resistance to aphids. The vigorous growth and flowering and high performance
of all lines of C. pulchra under outdoor trial conditions indicate that the species is an excellent candidate as a new bedding and landscape flowering annual. C. schumannii, on
the other hand possesses minimal potential as a new floriculture crop. The indeterminate growth habit, particularly outdoors, susceptibility to greenhouse pests, and susceptibility to intumescence injury make this an unlikely plant for introduction. In an official new crops research program, C. schumannii would be excluded from continuing evaluation at this point. However, the decision was made to include the species in continuing evaluation as a comparative species.
63 CHAPTER 3
PROPAGATIVE METHODS
3.1 Introduction
During the initial observation period of a potential new crop, the method and ease
of propagation are evaluated. This phase of the research is simply asking “Can this
species be propagated and by what means” (Armitage, 1986). Once propagation of a
species is deemed economically feasible then research related to production aspects can
commence.
Propagation by seed and vegetative cuttings are the two most popular and feasible
propagation methods for Cuphea species already in the floriculture trade. The seed
dispersal mechanism of Cuphea is the characteristic that makes it a unique genus in the
family Lythraceae (Graham, 1988). The floral tube and capsule wall split unilaterally along the dorsal side at maturity followed by emergence of the placenta, which exposes the seed for dispersal (Graham, 1988). It is this dehiscent nature of the flower/fruit that
has posed as the greatest obstacle for domestication of Cuphea as an oil seed crop due to
difficulty of seed harvest. From an ornamental horticulture perspective, this
characteristic is less of a concern. Both C. schumannii and C. pulchra are self-
incompatible species, requiring the aid of pollinators for viable seed production, or
64 sexual reproduction. Species of Cuphea, like many wild species, exhibits seed dormancy, which can seriously hinder experimentation in certain species by significantly extending the amount of time necessary to cycle generations (Knapp, 1990). Reported dormancy periods for species range from exceeding two years in Cuphea viscosissima populations to less than four weeks in certain populations of Cuphea lanceolata (Knapp,
1989; Knapp and Tagliani, 1989). The majority of the research conducted on Cuphea seed dormancy issues resulted from a national breeding and selection effort to develop the genus as an oil seed crop. Prior to intensive work being conducted on methods to break Cuphea seed dormancy, seed embryo excision provided the most reliable method of inducing uniform seed germination. Efforts were made to develop an alternative to this time consuming method (Widrlechner and Kovach, 2000). Work with C. viscosissima revealed that at least four factors influenced the release of seed dormancy:
1) time of storage; 2) temperature of storage; 3) light during germination; and 4) temperature alteration during germination (Widrlechner and Kovach, 2000).
Cutting propagation is another widely utilized technique for Cuphea species currently in ornamental production including C. hyssopifolia, C. llavea, and C. ignea.
This propagative method allows for a shorter production time than seed propagation.
Vegetative propagation also insures that offspring (or clones) are genetically and morphologically identical to the parent stock plant.
The need for rooting hormones to induce uniform rooting is dependent on the species. For many species, rooting compounds are not required for rooting but will accelerate root initiation, increase uniformity, and increase the number and quality of the roots produced (Dole and Wilkins, 2005). Because auxins are the family of endogenous
65 plant hormones responsible for root formation, they serve as the active ingredient found
in commercial rooting compounds. Indole-3-butyric acid (IAA) and naphthaleneacetic
acid (NAA) are two most common auxin analogs used.
The trials in this research set out to identify the most economically feasible
method of propagating C. pulchra and C. schumannii. The objectives of seed trials were
to: 1) determine if high and uniform rates of germination of C. pulchra and C.
schumannii seed can be induced without significant pre-treatment methods and 2)
determine if, like most other Cuphea species, seed dormancy can be reduced by removal
of the seed coat using an embryo excision method. The objectives of the vegetative
cutting rooting trials were to: 1) evaluate the effect of rooting hormone treatments on the
timing of root formation of C. pulchra and C. schumannii cuttings and 2) identify a
commercially available rooting hormone that produces the highest uniformity for each
species.
3.2 Materials and Methods
3.2.1 Germination Rates of Untreated Seed
Trial 1. Initial observation of C. pulchra and C. schumannii germination rates commenced on June 27, 2001. Seed used for this trial were from the initial seed lot provided by Dr. S.A. Graham. The C. schumannii seed was pooled from plants maintained at Kent State University from 1991 through 1999. Therefore, there was no way to be certain the duration of time the seed had been in dry storage. Seed of C. pulchra were collected by Dr. Graham in January 1998 in Bahia, Brazil. The approximate duration in room temperature dry storage of these seed was 3 years and 5
66 months. Fifty seeds of each species were sown into a 27.9 cm x 514.6 cm x 3.8 cm 162
cell plug tray (one seed per cell). The plug tray was placed in the mist house under
natural light. Six-second misting was facilitated by a bench- top misting system at a six
minute interval from 7:00-20:45 HR. Germination counts were conducted 7, 14, and 21
days after sowing.
Trial 2. The second trial to verify germination rates of C. schumannii and C. pulchra seed commenced on January 22, 2002. Seed used was collected during the 2001 outdoor trial and held in cold (refrigerated)/ dry conditions or room temperature/dry conditions for 6 months. Seeds received a 30-minute hot water pretreatment prior to being surface sown into 27.9 cm x 514.6 cm x 3.8 cm 162 cell plug tray (one seed per cell) using a soilless media (Metro-Mix 360, Scotts Co, Marysville, OH). Twenty seed from each storage treatment for both species were sown. Plug trays were placed under daytime misting (six-second duration every ten minutes from 7:00-17:00 HR) conditions.
Germination counts were conducted on 7, 14, and 21 days after sowing.
3.2.2 Seed Embryo Excision
Trial 1. C. pulchra and C. schumannii seed used in the first embryo excision trial initiated on September 25, 2003 had been in dry storage (at room temperature) for 12 months. Sixty seeds of both species were rinsed three times and soaked in distilled water overnight to soften the seed coat. The sixty seeds of each species were divided into groups of ten seeds. Three groups of ten seed were placed in three labeled plastic Petri on moistened germination-blotter paper. These three Petri dishes served as three control replicates. The USDA (Ames, Iowa) provided the protocol for Cuphea seed embryo
67 excision. All work to excise the embryo was performed under a dissecting microscope.
While holding the seed with forceps at the micropyle end of the seed, a scalpel was used
to cut along the opposite edge. The seed was gently removed from the seed coat using a
second pair of forceps. The excised seed was placed on moistened germination-blotter
paper in a plastic Petri dish. For C. schumannii, the three embryo excision treatment
replicates included 7,6, and 8 excised embryos respectively. C. pulchra embryo excision
replicates included 10, 7, and 6 embryos respectively. Each Petri dish was sealed using
Parafilm and placed in a 21ºC growth chamber under 24-hour low intensity fluorescent
light. Germination counts for excised and non-excised seed were conducted at 4, 7, 11,
14, and 21 days.
The percent germination data per control and treatment replicates was
transformed using the Arcsine square root transformation (Zar, 1974). Significance of
treatment effects on germination were determined by a one-way ANOVA. If the P value produced by the one-way ANOVA was <0.05, a Tukeys pairwise multiple comparison test was performed. Data analysis was performed using Sigma Stat statistical software
(Jandel Corp./SPSS, Chicago, IL).
Trial 2. Seed utilized for the second seed excision trial initiated on April 30, 2004 had been in dry storage at room temperature for 6 months. Seed was rinsed three times with distilled water and soaked for four hours to soften the seed coat. Excision protocol follows that outlined above. Excised and non-excised seed were place on moistened germination-blotter paper in plastic Petri dishes. C. schumannii control had 10 non-
excised seeds and the excised treatment had 12 excised seeds. C. pulchra control had 13
non-excised seed and 13 excised seeds as the treatment group. All seed was held under
68 low intensity fluorescent light at lab room temperature conditions. Germination counts were conducted at 4, 7, 10, 14, 17, and 21 days.
3.2.3 Acceleration of Cutting Rooting with Hormone Treatment
Trial 1. A preliminary trial using C. schumannii cuttings was conducted to determine which (if any) rooting hormone would enhance the number and time of root formation in comparison to un-treated cuttings. Commercially available rooting hormone powders used were Hormodin®-1 (0.1% indol-3-butyric acid), Hormodin®-3 (0.8% indol-
3-butyric acid), and Rootone® F (0.067% 1-napthaleneacetamide, 0.033% 2-methyl-1- napthaleneacetic acid, 0.013% 2 methyl-1-napthalmeactamide, and 0.057% indole-3- butyric acid). Cuttings were dipped in one of the three rooting powder treatments or left untreated as control and stuck into 13.3 cm x 13.3 cm x 5.9 cm packs (six cells per pack, eight packs per flat) using soilless media (Metro-Mix 360, Scotts Co., Marysville, OH).
Flats of cuttings were placed under daytime misting of six seconds at a six-minute interval from 7:00-20:45 HR. Digital images were used to capture visible difference between treatments on 5, 9, 14 and 19 days after sticking.
Trial 2. Both C. schumannii and C. pulchra “B” were the subjects of this rooting hormone trial. Only one rooting hormone, Hormodin®-1, was compared with untreated controls. Twenty-five cuttings of each species were dipped in rooting hormone powder and 25 cuttings of each species remained untreated. All cuttings were stuck into 13.3 cm x 13.3 cm x 5.9 cm packs (six cells per pack, eight packs per flat) using soilless media
(Metro-Mix 360, Scotts Co., Marysville, OH). Flats of cuttings were placed under daytime misting in the mist house. Five treated and five untreated cuttings for each
69 species were pulled on 7, 11, 14, 18 and 21 days after sticking and root number and average root length were recorded. Digital images were used to document differences between Hormodin®-1 treated and control cuttings.
3.3 Results and Discussion
3.3.1 Seed germination
The initial seed germination trial using seed provided by Dr. Shirley Graham yield poor results. After 21 days under daytime misting conditions only one C. schumannii seed germinated and no C. pulchra seeds germinated (data not shown). When under moist conditions, Cuphea seed releases seed hairs, which closely resemble fungal hyphae, from the epidermal cells of the seed coat (Figure 3.1). The purpose of these seed hairs is to anchor the seed to the growing substrate (Graham, 1988 and references cited therein). Sown seed of C. pulchra and C. schumannii produced these seed hairs within 1 hour of being sown and placed under misting conditions. However, because none of the seed germinated in the 21-day period, it can be assumed that the release of seed hairs does not signal seed viability or a break in dormancy.
Quite a few factors exist that may have caused such low (no) germination.
Firstly, the amount of time in storage after-ripening, particularly for C. schumannii seeds was indeterminate. The seed used was pooled from 1991-1999, meaning that seed could have been in storage 2 to 10 years prior to use in this trial. Based on the collection date,
C. pulchra seeds had been in storage for over three years. Secondly, the seeds were held under dry storage conditions at room temperature from the time of collection. However,
70
Figure 3.1. Seed hairs of a Cuphea schumannii seed. Hairs move out of the epidermal cells of the seed coat of Cuphea species after a short duration of exposure to water. The hair serve to anchor the seed to the Germination substrate.
based on germinability studies with C. viscosissima, which maintained high levels of
germination after four years of storage at room temperatures (Widrlechner and Kovach,
2000), one would have expected to see some, though perhaps low, rates of germination.
Thirdly, seeds were not pre-treated prior to sowing. Results of sowing without pre-
treatment indicated that C. schumannii and C. pulchra, like many other Cuphea species
may possess significant seed dormancy issues and mechanisms to break dormancy must
be employed for successful germination.
71 % Germination 7 day 14 day 21 day C. schumannii (cold/dry storage) 13.3 13.3 13.3 C. schumannii (room temp./dry storage) 0.0 0.0 75.0 C. pulchra (cold/dry storage) 0.0 25.0 37.5 C. pulchra (room temp./dry storage) 0.0 50.0 62.5
Table 3.1. Percent germination of Cuphea schumannii and Cuphea pulchra seed held under cold or room temperature storage for six months prior to sowing at 7, 14, and 21 days after sowing. Final percent germination data indicates that temperature of seed storage has an effect on the rate of germination for seeds of both species.
Germination of seed collected from plants that were part of the 2001 outdoor trial and
held under room temperature or cold storage for six months yielded better results than
that of the initial germination trial discussed above (Table 3.1). There were significant
differences in the seed used for this trial than that used in the first. Seed used in this trial had only been in storage for six months after-ripening under either room temperature dry storage or cold dry storage, and were soaked in hot water for 30 minutes prior to sowing.
After 21 days, regardless of storage conditions prior to sowing, 35% of C. schumannii
and 50% of C. pulchra seed had successfully germinated. This is by no means
considered a good rate of germination, yet illustrates a rate slightly improved from seed
2+ years after ripening. An interesting result is that for both species, there was a higher
rate of germination of seeds held under 6 months of dry, room temperature storage than
cold storage. For C. schumannii, germination rates of seed stored at room temperature or
cold storage prior to sowing were 75.0% and 13.3%, respectively and 62.5% and 37.5% 72 respectively for C. pulchra seed. For C. viscosissima seed, long-term, moist, cold-treated
seeds released dormancy faster than dry, room temperature storage (Widrlechner and
Kovach, 2000). Perhaps, for C. schumannii and C. pulchra seed held under cold treatment, the duration of storage (six months) was not long enough duration to evoke a more rapid release of dormancy. Because seed moisture content of the tested seed was not know it is not possible to conclude if, like C. vicsosissima, a higher seed moisture
content would significantly increase germination of C. schumannii or C. pulchra under
either temperature storage condition.
3.3.2 Seed Embryo Excision
Results of the seed germination trials indicate that C. schumannii and C. pulchra,
like many wild species of Cuphea, have significant seed dormancy issues. By employing an embryo excision technique it was confirmed that seed dormancy in these two species is the result of exogenous dormancy imposed by the seed coat (Figure 3.2). Results of the first seed excision trial indicated that removal of the seed coat accelerates the timing of germination and the percentage of germination over intact seeds of both Cuphea schumannii and Cuphea pulchra (Tables 3.2). After 21 days, 82.6% excised Cuphea pulchra seed had germinated whereas only 53.3% of non-excised seed had germinated.
Germination of excised Cuphea schumannii seed reached 50% by 21 days, whereas non- excised exhibited only 10.3% germination. For both C. pulchra and C. schumannii, the maximum rate of germination of excised seed was reached 7 days after initiation at
82.6% and 50% respectively. Germination of non-excised seed of both species increased gradually from day 7 to day 21.
73
Figure 3.2. Images of Cuphea pulchra seed with seed coat (left) and excised from seed coat (right).
Mean % Germination 4 days 7days 11 days 14 days 18 days 21 days C. pulchra excised 48.1 a 80.8 a 80.8 a 80.8 a 80.8 a 80.8 a C. pulchra non-excised 0.0 b 0.0 b 26.7 b 36.7 a 43.3 a 53.3 a
C. schumannii excised 54.2 a 63.7 a 63.7 a 63.7 a 63.7 a 63.7 a C. schumannii non-excised 0.0 b 3.3 b 3.3 b 3.3 b 6.7 b 10.0 b a,b Values followed by the same letter are not significantly different at P<0.05 (within species comparison only)
Table 3.2. Effect of seed embryo excision on mean percent germination of Cuphea pulchra and Cuphea schumannii.
The expected percentage of germination for excised seed is close to 100%.
Ungerminated excised seed at the end of the 21-day period was assumed not viable.
However, results of this embryo excision fell short of expected germination percentage. 74 The explanation for this lies in the seed extraction technique. Embryo excision is time
consuming and requires a degree of manual dexterity not needed for typical germination
tests (Widrlechner and Kovach, 2000). Damage to the cotyledons may not impair
germination and up to one-half of the cotyledon can be removed without affecting
germination. However, any damage caused to the embryo will affect germination.
The second seed embryo excision trial yielded results similar to the first trial (data
not shown). Germination of non-excised seed was further delayed in this trial, however
final germination percentage of non-excise and excised seed were comparable to
germination percentages of the first trial (data not shown).
3.3.3 Effect of Rooting Hormone on Root Development in C. schumannii Cuttings
The first rooting assay conducted evaluated the differences in Cuphea schumannii
cutting rooting when treated with commercial rooting compounds, Hormodin®-1,
Hormodin®-3, and Rootone® F. Digital images documented the difference between treatments and observations of rooting differences were noted. Fourteen days after cuttings were taken, difference between the treatments became apparent (Figure 3.3) .
Hormodin®-1 treated cuttings averaged approximately 42 root outgrowths, which averaged approximately 2 cm in length. Hormodin®-3 treated cuttings had very dense
root growth but all were less than 1 cm. Rootone® F treated cuttings exhibited only root
nodes, none had penetrated the periderm. Control cuttings were comparable to Rootone®
F treated cuttings. On day nineteen, it became clear that the Hormodin®-1 treated C.
schumannii cuttings had produced the uniform rooting and cuttings best prepared for
75
Figure 3.3. Root formation on Cuphea schumannii vegetative cuttings treated (from left to right) with Hormodin®-1, Hormodin®-3, Rootone®-F, and the untreated control 19 days after sticking. Cuttings treated with Hormodin®-1 produced the greatest quantity of high quality roots.
transplant. Hormodin®-3 treated plants had a dense outgrowth of roots but roots were
very stunted. Because Hormodin®-3 is a rooting compound typically reserved for use on
more woody plant material, the levels of exogenous auxin provided were significantly
higher than that required for the herbaceous C. schumannii stem cuttings. Rootone® F cuttings performed poorly overall with minimal, short root outgrowths. Control cuttings outperformed Rootone® F and Hormodin®-3 treated cuttings by averaging 10 root outgrowths per cutting at 1.5 cm long. Results of this assay identified Hormodin®-1 rooting compound with 0.1% indol-3-butyric acid as the best supplemental rooting compound candidate.
76 3.3.4 Effect of Hormodin®-1 on Root Development on C. schumannii and C. pulchra
Stem Cuttings
Results of the initial C. schumannii cutting rooting trial showed that use of
Hormodin®-1 (0.1% indol-3-butyric acid) produced the most significant and uniform root
formation on Cuphea schumannii cuttings. The second trail was set up to compare the
effect of Hormodin®-1 against untreated controls of C. schumannii and C. pulchra cuttings. After 21 days there was no significant difference (P<0.05) in the average number of roots or average length of roots between treated and untreated C. pulchra cuttings (Table 3.3). After a 21-day rooting period, C. pulchra did not produce enough high quality roots to be able to withstand transplant shock. Therefore, rooting hormone with 0.1% indol-3-butyric acid is not effective in accelerating root formation or increasing the number of roots produced in C. pulchra. Additional rooting studies are required to determine if a greater concentration of exogenous auxin or different active ingredients could accelerate and increase rooting of C. pulchra.
In parallel to the results of the initial rooting trial, after 21 days, C. schumannii cuttings treated with Hormodin®-1 had a greater average number of roots per cutting than
untreated cuttings (41.8 compared to 22.3) and a slightly higher average root length than
untreated cuttings (4.7 cm compared with 3.7 cm). However, the relationship between
the treated cuttings and the controls is quite variable through day 14 when treated cuttings
begin to show an increase in the number of roots. The drastically lower average number
of roots on untreated cuttings observed on day 18 cannot be explained.
77
day 7 day 11 day 14 day 18 day 21 Avg. Length Avg. Length Avg. Length Avg. Length Avg. Length Avg. # roots (cm) Avg. # roots (cm) Avg. # roots (cm) Avg. # roots (cm) Avg. # roots (cm) C. schumannii 0.0 < 1cm 3.0 < 1cm 29.0 < 1cm 32.8 2.8 41.8 4.7 Hormodin-1 treated
78 C. schumannii control 1.0 < 1cm 4.7 < 1cm 20.7 <1 cm 5.0 1.7 22.3 3.7 C. pulchra Hormodin-1 0.0 0.0 3.5 < 1cm 5.5 < 1cm 3.5 2.3 3.7 3.7 treated
C. pulchra control 0.0 0.0 0.0 0.0 1.5 < 1cm 2.3 2.3 3.3 2.2
Table 3.3. Effects of exogenous rooting hormone (Hormodin®-1) on the average root number and average length of roots formed on vegetative cuttings of Cuphea schumannii and Cuphea pulchra at 7, 11, 14, 18, and 21 days after sticking.
3.4 Conclusions
Issues of seed dormancy pose a significant barrier to experimentation with C. pulchra and C. schumannii. Seed embryo excision is a successful technique to eliminate dormancy but is not an economically feasible technique for large-scale production. Like many of the Cuphea species that are currently commercially available, the most economically feasible method of propagating C. pulchra and C. schumannii is by vegetative stem cutting. The use of a 0.1% indol-3-butyric acid containing rooting compound slightly accelerated root formation on C. schumannii cuttings for plant material ready for transplant in approximately 19 days. Additional research is necessary to identify the concentration of exogenous auxin that will accelerate the root formation on
C. pulchra cuttings. This research would indicate that with or without the use of rooting compounds rooting time of C. pulchra cuttings for transplant would likely exceed 21 days.
79 CHAPTER 4
PRELIMINARY INVESTIGATION OF THE EFFECTS OF TEMPERATURE ON
GROWTH AND DEVELOPMENT
4.1 Introduction
The temperature at which a particular crop is grown can significantly impact plant growth and development. Each plant species has an optimum growing temperature range, one in which the best quality plants are produced in the least amount of time.
Each plant species also has a tolerable temperature range, one in which plants will grow.
However plant quality and production time are significantly affected in relation to those plants grown under optimal conditions.
Temperature is an environmental characteristic that can be altered to influence growth and development of crops grown in controlled environments. Research to find practical ways of regulating plant growth by utilizing alterations in growth temperature to induce wanted variations in morphological characteristics of greenhouse crops is ongoing. There are three primary temperature manipulation mechanisms: 1) DIF, which is the control of plant growth through manipulation of the difference between day temperature and night temperature; 2) DROP, which is the lowering of the growing temperature during the first 2-4 hours of the day just prior to dawn; 3) average daily
80 temperature (ADT), which is the temperature to which a plant is exposed over a 24-hour
period calculated as an average of temperatures measured each hour.
DIF strongly influences plant height and internode length of many plant species
(Myster and Moe, 1995). Through study with lily, differences in stem elongation were
found to be due to differences in cell elongation, which increased as DIF increased from a
negative value to a positive value (Erwin et al., 1991). Internode elongation increases
more as DIF increases from zero to a positive value than from a negative value to zero
(Heins and Erwin, 1990). DIF can also influence flower initiation and development and response varies between long day and short-day plants. DIF has only a small influence on flower initiation and development of long-day plants (Myster and Moe, 1995).
However, in short-day plants, such as poinsettia, negative DIF significantly delays flowering (Moe et al., 1992).
Stem elongation of some greenhouse crops is sensitive to short periods of temperature drop or temperature increase (Myster and Moe, 1995). The effect of this short fluctuation is most effective 2-4 hours before dawn. A regime of increased temperature during that time window will result in plants with and increase in stem elongation. Conversely, if the temperature regime involves a decrease in temperature during the 2-4 hour period, plants will exhibit a reduction in height and internode length, similar to plant response to negative DIF
Altering the ADT to which greenhouse crops are exposed under controlled growing conditions can influence internode elongation in some species (Myster and Moe,
1995). Average daily temperature can also affect leaf unfolding or expansion, whereas
81 DIF does not (Moe and Heins, 1990). Studies with the perennial Campanula show that an increase in ADT enhances flowering in long-day plants (Moe, 1993).
The effect of temperature on plant development rate is important to accurately time crop production (Clough et al., 2001). No information exists regarding effect of production temperature on C. pulchra or C. schumannii. As species native to Central and
South America they have adapted to warmer average yearly temperatures than that of central Ohio Zone 5. Under native climatic conditions, C. pulchra is accustomed to growth under average yearly temperatures of approximately 25ºC (based on average monthly temperatures from 1961 through 1990 for Aracaju, Brazil 10.92ºS 37.00ºW, www.worldclimate.com). Similarly, C. schumannii native growing temperatures average approximately 25ºC (based on average monthly temperatures from 1947 through 1983 for
Ojitlan, Oaxaca, Mexico, www.worldclimate.com). It is important that the optimum growing temperature range for both species under greenhouse production conditions be explored to develop cultural information pertinent to the production of the best quality plant in the shortest amount of time.
The objectives of this study were to evaluate the growth and development of C. pulchra and C. schumannii under three controlled temperature regimes. Plant growth and development parameters of particular interest were plant height, bud set, flowering, and lateral branching. A secondary objective of this study was to elucidate the optimal and tolerable temperature ranges for the two species.
82 4.2 Materials and Methods
Plant Culture
Four week-old rooted vegetative stem cuttings of C. pulchra and C. schumannii were transplanted into 15.24 cm diameter standard pots using MetroMix 360 (Scotts Co.,
Marysville, OH) soilless media. Under experimental conditions, pots were watered by hand as needed and fertilized once per week with 200 mg N L-1 of 20-10-20 Peters
complete fertilizer (Scotts Co., Marysville, OH). Each pot was held in a plastic saucer to
contain draining water.
Growth Chamber Treatment Conditions
Seven plants of each species were maintained under one of three temperature
regimes in a Conviron (Winnipeg, Canada) growth chamber. Growth chamber
temperatures were set to 13ºC/10ºC day/night (low), 21ºC/18ºC day/night (mid), and
29ºC/26ºC day/night (high). All chambers were programmed for a 13 hour light period provided by both fluorescent and incandescent light bulbs. Initial light intensity in the chambers was approximately 260 µmol m-2 s-1. During the third replicate trial,
cumulative photosynthetic photon flux (PFF), temperature, and relative humidity were
monitored in all three chambers using appropriate sensors and data loggers (Spectrum
Technologies, Plainfield, IL) (Table 4.1).
83 Average 6 Week Hourly Average Cumlative PPF Temperature Hourly RH Treatment (mol m-2s-1) (ºC) (%) low 0.12 13.1 82.5 mid 0.14 17.9 66.8 high 0.11 28.1 58.8
Table 4.1. Cumulative photosynthetic photon flux (PPF), average hourly temperature, and average hourly relative humidity environmental data collected from growth chambers maintaining the experimental temperature regimes of 13ºC/10ºC (low), 21ºC/18ºC (mid), and 29ºC/26ºC (high). Data collected by data loggers in each growth chamber during the third and final replicate trial.
Evaluation and Analysis
C. pulchra and C. schumannii plants were held under experimental temperature conditions for six weeks. Initial height and leaf number were recorded. Final data collected at six weeks after initiation included final height, leaf number, node number, number of lateral branches (longer than 2 cm), number of visible flower buds, number of mature flowers (mouth of hypanthium open), and pot media temperature prior to removal from the growth chamber. Change in plant height and average internode length was calculated. All plants were destructively harvested, shoot and root were separated, dried for 72 hours in a drying oven, and dry weights recorded. Root:shoot dry mass ratio was calculated. Due to space constraints in the growth chambers the experiment was conducted three times to increase replication (in time) for analysis. At the conclusion of each replicate trial, digital images were taken to document the difference in plant appearance between treatments.
84 Significance of treatment effects on change in plant height, leaf number, node number, average internode length, number of lateral branches, number of flower buds, number of flowers, soil temperature, shoot dry weight, root dry weight, and root:shoot ratio was determined by analysis of variance (ANOVA). If the P value produced by the one-way ANOVA was <0.05, a Tukeys pairwise multiple comparison test was performed. The statistical program used was SigmaStat software program (Jandel
Corp./SPSS, Chicago, IL ).
4.3 Results and Discussion
Temperature had a very dramatic effect on the growth and development of both C. pulchra and C. schumannii. For C. pulchra plants there were statistically significant
(P<0.05) differences between all three temperature regime treatments for plant parameters; change in height, number of leaves, number of nodes, and average internode length increased as temperature increased (Table 4.2). Lateral branching of C. pulchra was significantly different between plants maintained under low temperatures and those under mid and high temperatures. Visible bud formation was significant between mid and high temperature treated plants. Often, when plant species is grown under temperatures that fall at the high end of its tolerable temperature range, flower initiation and development are delayed (Clough et al., 2001). For C. pulchra six weeks after initiation, plants grown under the mid temperature range (21ºC) had had produced significantly more visible flower buds than those maintained under a daytime temperature regime 8ºC higher.
85 Change in height and the number of nodes were parameters that were significantly
different (P<0.05) between all three temperature treatment for C. schumannii (Table 4.3).
Leaf number, average internode length, and lateral branching were significant between
low temperature treated plants and mid and high temperature treated plants. Visible bud
formation and number of mature flowers was not significant.
Destructive harvest of all plants was conducted to determine temperature affects
on growth of aerial portions of the plant (stem and leaves) and roots. The root:shoot ratio
was calculated to evaluate if plant allocation to root or shoot growth varies significantly
between the three temperature treatments. C. pulchra plants maintained under high
temperature conditions had significantly higher (P<0.05) shoot dry mass than those grown under mid or low temperature conditions (Table 4.4). Root dry weight was significant only between high temperature and low temperature conditions. However, the root:shoot ratio decreased significantly as the temperature increased indicating that as temperature increases, the plant allocates more resources to the development of the plant shoot than the root. Shoot dry weights of C. schumannii grown under low temperature conditions were significantly lower (P<0.05) than those plants grown under mid or high temperature regimes (Table 4.5). C. schumannii plants maintained under the mid temperature treatment exhibited a significantly higher root dry weights than those maintained under low or high temperature conditions. Like C. pulchra, as temperatures increased so to did the root:shoot ratio with a significant decrease in the ratio when temperatures increased 8-10ºC from the mid temperature treatment to the high temperature treatment.
86
Average # Lateral Pot Media Temperature Change in # Leaves per # Nodes on Internode Branches # Flower # Mature Temperature Treatment Height (cm) Plant Main Stem * Length (cm) (over 2cm) buds Flowers (ºC) a a a a a ab a a 13ºC / 10ºC 6.9 ± 1.2 17.6 ± 2.4 6.4 ± 0.4 1.7 ± 0.1 5.4 ± 0.9 5.9 ± 2.0 0 ± 0.0 15.0 ± 0.5 87 21ºC / 18ºC 17.8 ± 1.8 b 67.2 ± 6.3 b 10.1 ± 0.6 b 2.1 ± 1.0 b 10.6 ± 1.0 b 17.7 ± 4.7 a 1.3 ± 0.5 b 19.1 ± 0.7 b
29ºC / 26ºC 34.2 ± 1.9 c 125.6 ± 9.8 c 12.9 ±0.4 c 2.9± 1.1 c 9.8 ± 1.1 b 0.9 ± 0.6 b 0.4 ± 0.2 ab 29.1 ± 0.7 c a,b,c, Values followed by the same letters are not statistically different at P <0.05 * The main stem was the stem used to measure initial and final height.
Table 4.2. Treatment effects on parameters of Cuphea pulchra plants maintained under 13ºC/10ºC (low), 21ºC/18ºC (mid), or 29ºC/26ºC (high) growth chamber temperature regimes. Values represent the mean of 21 plants (three replicates of 7 plants each) ± standard error. Change in height was calculated by subtracting the initial plant height from the final plant height. Average internode length was calculated by dividing the height of the main stem by the number of nodes on that stem.
Average # Lateral Pot Media Temperature Change in # Leaves per # Nodes on Internode Branches # Flower # Mature Temperature Treatment Height (cm) Plant Main Stem * Length (cm) (over 2cm) buds Flowers (ºC) a a a a a a a a 13ºC / 10ºC 10.5 ± 1.0 18.4 ± 1.6 8.2 ± 0.3 1.9 ± 0.2 1.9 ± 0.5 4.4 ± 0.8 0.0 ± 0.0 14.6 ± 0.5
b b b b b a ba b 88 21ºC / 18ºC 29.5 ± 2.6 47.1 ± 5.7 11.1 ± 0.6 3.0± 0.2 5.9 ± 0.5 22.6 ± 5.3 1.1 ± 0.5 18.7 ± 0.7
29ºC / 26ºC 46.8 ± 3.2 c 76.0 ± 10.5 b 15.7 ± 1.4 c 3.5 ± 0.2 b 7.6 ± 0.9 b 22.1 ± 6.3 a 1 ± 0.4 b 29.4± 0.8 c a,b,c, Values followed by the same letters are not statistically different at P <0.05 * The main stem was the stem used to measure initial and final height.
Table 4.3. Treatment effects on parameters of Cuphea schumannii plants maintained under 13ºC/10ºC (low), 21ºC/18ºC (mid), or 29ºC/26ºC (high) growth chamber temperature regimes. Values represent the mean of 21 plants (three replicates of 7 plants each) ± standard error. Change in height was calculated by subtracting the initial plant height from the final plant height. Average internode length was calculated by dividing the height of the main stem by the number of nodes on that stem.
Temperature Shoot Dry Root Dry Root: Shoot Treatment Weight (g) Wei ght (g) Ratio 13ºC / 10ºC 0.4 ± 0.1a 0.2 ± 0.0a 0.5 ± 0.1a
21ºC / 18ºC 1.6 ± 0.3a 0.3 ± 0.0ab 0.2 ± 0.0b
29ºC / 26ºC 3.6 ± 0.3b 0.4 ± 0.1b 0.1 ± 0.0c a,b,c, Values followed by the same letters are not statistically different at P <0.05
Table 4.4. Treatment effects on shoot dry weight, root dry weight, and root:shoot ratio of Cuphea pulchra plants maintained under 13ºC/10ºC (low), 21ºC/18ºC (mid), or 29ºC/26ºC (high) growth chamber temperature regimes. Values are the mean of 14 plants (two replicates with 7 plants each) ± standard error.
Temperature Shoot Dry Root Dry Root: Shoot Treatment Wei ght (g) Wei ght (g) Ratio 13ºC / 10ºC 2.4 ± 0.5a 0.6 ± 0.1a 0.2 ± 0.0a
21ºC / 18ºC 10.2 ± 1.1b 1.9 ± 0.1b 0.2 ± 0.0a
29ºC / 26ºC 9.4 ± 0.7b 1.2 ± 0.1a 0.1 ± 0.0 b a,b, Values followed by the same letters are not statistically different at P <0.05
Table 4.5. Treatment effects on shoot dry weight, root dry weight, and root:shoot ratio of Cuphea schumannii plants maintained under 13ºC/10ºC (low), 21ºC/18ºC (mid), or 29ºC/26ºC (high) growth chamber temperature regimes. Values are the mean of 14 plants (two replicates with 7 plants each) ± standard error.
Although plant growth parameters between temperature treatments are significantly different, the values do not provide an accurate evaluation of plant appearance. Figure 4.1 illustrates the difference in plant appearance between C. pulchra plants. Maintenance under the mid (21ºC daytime temperature) range produced the 89 Low Mid High
Figure 4.1. Effect of temperature on growth and development of Cuphea pulchra. Plants shown above (from left to right) were held under 13ºC/10ºC (low), 21ºC/18ºC (mid), or 29ºC/26ºC (high) temperature regimes in growth chambers to evaluate temperature effects on plant growth, development, and appearance after 6 weeks.
best quality plants when compared with those plants maintained under the low (13ºC daytime temperature) or the high (29ºC daytime temperature) temperature treatments.
Plants at the mid temperature range exhibited a compact growth habit, uniform lateral branching, healthy foliage color, and produced the most visible buds and mature flowers in the six week study period. Cuphea pulchra held under low temperature conditions exhibited stress symptoms. A noticeable response to the low temperatures was the reddish-purple pigmentation of the newly developing leaves (Figure 4.2). This coloration was due to over production of anthocyanin, a water-soluble vacuolar pigment.
90
Figure 4.2. Low temperature effects on anthocyanin production in foliage of C. pulchra. C. pulchra maintained under 13ºC/10ºC temperatures exhibited reddish-purple coloration of leaves, particularly new growth. This accumulation of anthocyanin was the plant response to low temperature stress. Tissue nutrient analysis eliminated the possibility that anthocyanin accumulation was symptomatic of sulfur, phosphorus, or nitrogen deficiency.
Accumulation of anthocyanin can be an indicator of a phosphorus, sulfur, or nitrogen deficiency (Taiz and Zeiger, 1998). Results of tissue analysis, however, indicated that there was no significant difference in the levels of these nutrients between plants maintained under the three temperature treatments (data not shown). Unlike pigmentation in flowers and fruits, anthocyanin accumulation in leaves is due mainly to environmental stress (Harbone and Grayer, 1988). Low temperatures have been shown to induce increased anthocyanin synthesis in leaves of the woody subtropical cocoplum
(Nissim-Levi et al., 2003), maize seedlings (Christie et al., 1994), and smoke bush
91 (Oren-Shamir and Nissim-Levi, 1997). This suggests that the reddish-purple coloration
of Cuphea pulchra of low temperature treated plants was caused by an accumulation of
anthocyanin in response to colder temperatures.
Appearance of C. schumannii was also noticeably different between the three
temperature treatments (Figure 4.3). C. schumannii plants maintained under the mid
temperature regime were of better quality than those under low temperatures and similar
quality to those under high temperatures but shorter in stature. As indicated in Chapter 2,
C. schumannii, is prone to developing leaf intumescence under controlled environmental
conditions. It is worthy to note that leaf intumescence did not develop under growth
chamber conditions and was not influenced by temperature.
low mid high
Figure 4.3. Effect of temperature on growth and development of Cuphea schumannii. Plants shown above (from left to right) were held under 13ºC/10ºC (low), 21ºC/18ºC (mid), or 29ºC/26ºC (high) temperature regimes in growth chambers to evaluate temperature effects on plant growth, development, and appearance after 6 weeks. 92 4.4 Conclusions
Results of this study indicate that growth and development of both C. pulchra and
C. schumannii are significantly influenced by the temperature at which they are grown.
As species native to climates with yearly temperatures averaging 25ºC it was not unexpected to observe the significant stress response to low production temperatures.
Although this study was not designed to determine the specific optimum or tolerable temperature ranges it was a successful preliminary investigation that defined temperature ranges for further investigation. For C. pulchra, the 21ºC/18ºC mid temperature treatment produced the highest quality plants with compact growth, healthy foliage, and significant visible bud formation in a six week time period. This temperature can certainly be assumed to fall within the optimal temperature range for growth and development of C. pulchra. The 13ºC/10ºC low temperature treatment would fall outside low tolerable temperature. Plant performance under these temperatures was poor, producing significantly fewer leaves, lateral branches, and flower buds. Additionally, these low temperatures had a significant negative impact on vegetative vigor and color of
C. pulchra. Based on performance of C. pulchra under 29ºC/26ºC high temperature conditions, one may conclude that this temperature is within the high tolerable temperature range. Although plants were attractive under these conditions, the significantly lower bud set indicates a delay in development, which would exclude this temperature from falling within the optimum temperature range for C. pulchra. Based on climatic data for areas to which C. pulchra is native, the average high temperature during the year is approximately 27ºC that occurs during the months of January, February, and
March. Interestingly, these warmer months proceed two months (April and May) when
93 there are fewer flowers (S.A. Graham, personal communication). This would help to
explain why C. pulchra growing under controlled temperature averaging approximately
28ºC over a six week period had a lower bud set. Because of C. schumannii lack of
potential as a successful ornamental crop, determining optimum and tolerable
temperature ranges is not an immediate necessity. However, based on performance of the
species under the experimental temperature conditions, it can be concluded that the
temperature ranges for C. schumannii would closely parallel those for C. pulchra.
94 CHAPTER 5
METHODS OF GROWTH REGULATION
5.1. Introduction
Plant growth regulation can be defined as any process or chemical that is employed to produce a specific type of growth response (Dole and Wilkins, 2005).
Methods of plant growth regulation for greenhouse crops are typically used to control vegetative growth. Without some form of growth control, many species or cultivars will develop extended internodes and weak branches, which contribute to lower leaf loss.
Many ornamental plants undergo growth retardation treatments to produce compact, healthier plants that can better withstand handling and shipping and thus have higher marketability.
Methods of plant growth regulation can be divided into two broad categories: chemical or non-chemical methods. Chemical plant growth retardants or plant growth regulators (PGRs) are synthetic compounds used to reduce the shoot length of plants in a desired way without negatively altering plant development (Rademacher, 2000). These
PGRs act by reducing the rate of cell division and cell elongation. Chemical growth regulators can be classified as either an ethylene-releasing compound or inhibitors of gibberellin (GA) biosynthesis (Rademacher, 2000). This study focused on the
95 effectiveness of three PGRs classified as inhibitors of gibberellin biosynthesis:
chlormequat, paclobutrazol, and daminozide. These specific chemicals represent three of
the four groups of GA biosynthesis inhibitors. Chlormequat is an onium-type compound,
which blocks GA biosynthesis directly before ent-kaurene (Rademacher, 2000).
Paclobutrazol is a compound with a nitrogen-containing heterocycle, which acts as an inhibitor of monooxygenase activity catalyzing the step between ent-kaurene and ent- kaurenoic acid (Rademacher, 2000). Daminozide is a structural mimic of 2-oxoglutaric acid, which blocks GA formation as an inhibitor of 2-oxoglutarate-dependent dioxygenases (Rademacher, 2000). The fourth group of GA biosynthesis inhibitors, which is not represented in this study, is the 16, 17-dihydro-GAs. This group of compounds inhibits dioxygenases that catalyze the late stages of gibberellin metabolism
(Rademacher, 2000).
The effectiveness of a PGR will be dependent on the plant species due to varying levels of sensitivity. For common greenhouse crops, information is available as to what commercially available PGR and at what concentration is most effective for growth regulation. However, there is no literature on the use of chemical growth retardants in the production of C. pulchra or C. schumannii. For a species for which no information exists regarding chemical growth control, it is necessary to conduct trials to determine how to achieve the best control with the least amount of chemical while still producing a high quality plant.
Despite high effectiveness of PGR, alternative, non-chemical methods of plant growth regulation are needed in light of the rising concerns of environmental pollution from greenhouse run-off. Several non-chemical methods of height regulation have been
96 employed in horticulture crop production including genetic selection, mechanical
conditioning, temperature management, and light quality manipulation (Dole and
Wilkins, 1999; Rajapakse et al. and references cited therein, 1999). Of these methods, greenhouse temperature manipulation has been the only method that has been widely applied in commercial production (Clifford et al., 2004). There are two practices currently in use that employ altering the diurnal temperatures to control plant growth: 1) the difference (DIF) between day and night temperatures particularly when night temperatures are maintained higher than the day temperatures; 2) a temperature drop for
2-4 hours before sunrise (Myster and Moe and references cited therein, 1995; Clifford et al., 2004).
Manipulation of the spectral composition of light is another promising non- chemical alternative to limiting plant growth. Considerable work with herbaceous ornamental crops such as poinsettias (Clifford et al., 2004), chrysanthemums (Oyaert et al., 1999; McMahon, 1999), and potted miniature roses (McMahon and Kelly, 1990), have shown that growth under spectral filters that reduce the transmission of far-red light results in reduced internode elongation and plant height.
Plant leaves absorb most red light (600-700nm) but reflect most far-red light
(700-800nm). It is the ratio of red to far-red (R: FR) light that the plants are able to detect and help them adjust to the environment. Phytochrome is a red/far-red reversible pigment, which senses the alterations in the light environment and is required for plant developmental responses to red and far-red light. The Pr form which absorbs red light and the Pfr form which absorbs far-red light are photointerconvertible, red light converts
Pr to Pfr and far-red light converting Pfr back to Pr (Vierstra, 1993). It is the ratio
97 between these two forms that signals the plant to make appropriate growth adjustments.
Under conditions where plants are growing under a plant canopy or are growing in close
proximity to on another, the red light available is reduced to a much greater extent than
the far-red light, thus reducing the R:FR (increasing the Pr:Pfr of the plant). This will
cause many plant to respond by increasing elongation growth, a response known as the
shade-avoidance response (Aphalo et al., 1999). The use of spectral filters works on the
premise that plant growth can be altered by manipulating the spectral quality of light to
which plants are exposed.
Early work with copper sulphate liquid spectral filters, which selectively excluded
FR, was successful in controlling plant extension growth. However, these liquid filters
can only be used in greenhouses that have been specifically designed for the purpose.
High capital costs and health and safety issues surrounding the handling of the liquid
filters makes their use impractical for commercial production (Rajapakse et al., 1999). In
the past decade, lightweight, flexible, polyethylene, plastic filters have been developed
that limit the transmission of FR and have been shown to successfully reduce extension
growth of some ornamental crops (Oyaert et al., 1999; McMahon, 1999; Clifford et al.,
2004).
This portion of the research set out to begin developing information for the
practical control of growth of Cuphea schumannii and Cuphea pulchra under production conditions. The study was developed to evaluate the species response to chemical methods of height control and non-chemical method of height control using photoselective filters. The objective of the chemical growth regulator study was to determine the effectiveness of three concentrations of commercially available chemical
98 PGRs Bonzi® (paclobutrazol), B-Nine® (daminozide), and Cycocel® (chlormequat) in
controlling the growth of Cuphea schumannii and Cuphea pulchra. The objective of the
FR filter study was to evaluate if use plastic photoselective filters are an effective non- chemical method to reduce elongation of C. schumannii or C. pulchra.
5.2. Materials and Methods
5.2.1 Chemical Growth Retardants
Cuphea schumannii
The chemical growth regulator study for C. schumannii was initiated on October
14, 2003 under greenhouse conditions. Plants were maintained under natural photoperiods. When natural light intensity fell below 300Wm-2, supplemental lighting
from 1000W metal halide lamps (Hydrofarm, Petaluma, CA) was provided from sun up
to 16:00 HR. Plants were watered by hand daily and fertilized twice a week with 200 N
L-1 of 20-10-20 Peters Complete fertilizer (Scotts Co., Marysville, OH). Temperature,
relative humidity, and photosynthetic photon flux of the greenhouse were monitored
using appropriate sensors and data loggers (Spectrum Technologies, Plainfield, IL)
(Table 5.1)
Sixty, four-week old rooted cuttings, planted in soilless media (Metro-Mix 360,
Scotts Co., Marysville, OH) in 15.24 cm diameter plastic pots, were evaluated for initial
height, number of nodes, and number of expanded leaves. Plants were labeled and
divided into treatment groups of 5 (the control group was comprised of 7 plants) and
arranged in a randomized block design. Treatment groups were B-Nine® (Uniroyal
Chemical, Middlebury, CT), 2500 ppm, 3750 ppm, and 5000 ppm; Cycocel ®
99
Average Average Cumulative PFF Temperature Relative Species Study Period (mol m-2) (ºC) Humidity (%) C. schumannii 5 weeks 0.49 24.2 38.1 C. pulchra 8 weeks 0.64 21.5 40.0
Table 5.1. Cumulative photosynthetic photon flux, average temperature, and average relative humidity of greenhouse growing conditions for the experimental periods of 5 and 8 weeks for C. schumannii and C. pulchra respectively.
(Olympic Horticultural Products, Mainland, PA)) 800 ppm, 1250 ppm, and 2000 ppm;
Bonzi® (Uniroyal Chemical, Middlebury, CT)10 ppm, 25 ppm, and 40 ppm; and manual
pinch leaving 5 to 6 nodes. The concentrations of B-Nine®, Cycocel®, and Bonzi®
chemical growth regulators used in this study were determined by previously conducted
pre-trials. Using hand-held spray bottles, each plant was spray treated at initiation with
30 mL of its respective chemical concentration. Growth regulator was applied to all
visible leaf and stem surfaces. Pinched and control plants were spray treated with 30 mL
of tap water to maintain foliar moisture consistent with the chemically treated plants.
Plants were given a second 30 mL treatment application when new growth began to
elongate at two weeks after initiation. The study was concluded at five weeks. Final
height, diameter, number of nodes, number and length of lateral branches (longer than
100 2 cm), and number of leaves were recorded. Average internode length and average
lateral branch lengths were calculated. Digital images were taken to document visible
differences between treatments and the control plants.
An evaluation was designed to gain feedback on the quality of plants produced as
a result of the various treatments. A panel of 13 people (faculty, staff, and students of the Department of Horticulture and Crop Science, OSU) was asked to evaluate foliage color, vegetative vigor, and plant elongation for each of the treatment groups. Foliage color and vegetative vigor were rated on a scale of 1 to 5 (1=poor - 5= excellent). Plant elongation was also rated on a scale of 1 to 5 (1=excessively stunted - 3= good control -
5= excessively leggy). Evaluators were also asked to indicate the one treatment group that they believed to have the best overall appearance. The evaluation sheet also provided space for additional comments.
Following the evaluation, three plants from each group were destructively harvested. Leaf area (Li-Cor 3100 leaf area meter, Lincoln, NE) and collective dry weight (all three plants) was recorded.
Plant parameter data collected for the chemical growth regulator study was analyzed by analysis of variance to compare treatment effects. If the P value produced by
the one-way ANOVA was <0.05, a Tukeys pairwise comparison test against the control
was performed. Data analysis was performed using Sigma Stat statistical software
(Jandel Corp. /SPSS, Chicago, IL).
101 Cuphea pulchra
The chemical growth regulator study for C. pulchra was initiated on December 1,
2003. All greenhouse conditions were the same as those for the C. schumannii trial.
Average temperature, average relative humidity, and cumulative photosynthetic photon
flux of the greenhouse during this eight week experimental period can be found in Table
5.1.
Fifty-five, five-week-old C. pulchra “B” cuttings, planted in soilless media
(Metro-Mix 360, Scotts Co., Marysville, OH) in 15.24 cm diameter plastic pots, were
measured for initial height, number of nodes, and number of expanded leaves. Plants
were labeled and divided into treatment groups of five and arranged in a randomized
block design. The chemical growth regulators and concentrations used (determined by pre-trials) were the same as those used for the C. schumannii study. Designated plants
were pinched at the outset of the study leaving four nodes. Using hand-held spray
bottles, each plant was spray treated to cover all visible stem and leaf surfaces at
initiation with 20 mL of treatment PGR solution. Pinched and control plants were spray
treated with 20 mL of tap water. Plants were given a second 20 mL treatment application
when new growth began to elongate at 2.5 weeks after initiation and at six weeks after
initiation. The study was concluded after eight weeks. Final height, diameter, number of
nodes, number and length of lateral branches (longer than 2 cm), and number of leaves
were recorded. Average internode length and average lateral branch lengths were
calculated. Digital images were taken to document visible differences between
treatments and the control plants.
102 An evaluation of C. pulchra plants was conducted in the same manner as that
described for C. schumannii. Nine people participated in the evaluation. Following the
evaluation, three plants from each group were destructively harvested. Leaf areas (Li-
Cor 3100 leaf area meter, Lincoln, NE) and dry weights were recorded.
Data was analyzed by analysis of variance. If the P value produced by the one-
way ANOVA was <0.05, a Tukeys pairwise comparison test against the control was
performed. Data analysis was performed using Sigma Stat statistical software (Jandel
Corp. /SPSS, Chicago, IL).
5.2.2 Photoselective Filters
The first trial study to evaluate the effectiveness of far-red absorbing films on
height reduction of C. schumannii and C. pulchra was initiated on March 27, 2002. The
study was conducted under greenhouse conditions with a 16 hour photoperiod maintained
by supplemental lighting from 1000 W metal halide lamps (Hydrofarm, Petaluma, CA).
When the natural light intensity fell below 200 W m-2, the supplemental lighting provided a PPF of 100 µmol m-2. Two 120 cm x 60 cm x 60 cm frames were constructed using
2.5cm diameter PVC piping. One frame was draped with a double layer of YXE-10
polyethylene far red absorbing film (Mitsui Chemical Co., Japan) and the second frame
was draped with a double layer of 2mil polyethylene neutral density film (Figure 5.1).
The frames were placed in the middle of the second and fourth benches in a greenhouse
with five benches.
103
Figure 5.1. Experimental chambers used to evaluate plant growth under far-red absorbing films.
Sixteen, four-week-old cuttings of both Cuphea schumannii and Cuphea pulchra were transplanted into 15.24 cm diameter plastic pots one week before initiating the study. Initial measurements of the plants included plant height, number of nodes, and number of expanded leaves. Four plants of each species were randomly selected and placed under far-red absorbing film, neutral density film, or open bench (no film control) conditions. The photosynthetic photon flux (PPF) under all three conditions was monitored by use of a quantum light sensor and data logger (Spectrum Technologies,
Plainfield, IL) (Table 5.2). Plants were watered by hand daily and fertilized twice per week. The study was concluded after four weeks. The height, number of nodes, number
104 Trial 1 Trial 2 Average Cumulative PPF Cumlative PPF Temperature (mol m-2)R:FR (µmol m-2) (mol m-2) (ºC) FR Filter 0.08 1.7 0.11 25.4 Clear Filter 0.08 1.2 - - Open Bench 0.11 1.3 0.13 24.2
Table 5.2. Cumulative photosynthetic photon flux (PPF) for photoselective filter trial 1 (four week period March 27, 2002- April 24, 2002) and cumulative PPF and average hourly temperature for trial 2 (four week period July 22, 2002 – August 19, 2002). R: FR readings were taking on a sunny mid-morning in July. Cumulative PPF during the second trial and average temperature was not recorded under clear filter conditions.
of expanded leaves, number of lateral branches, and number of visible buds and flowers
was recorded. Changes in height and average internode length were calculated. Digital
images were taken to document visible differences between treatment groups.
This study was repeated beginning July 22, 2002. Protocol follows that described
above. In addition, temperature under the far-red filter and open bench treatment
conditions was monitored using data loggers (Spectrum Technologies, Plainfield, IL)
(Table 5.2). The ratio of R: FR was measured for each of the three treatment conditions
(Table 5.2). Measurements were taken mid-morning on July 20 using a Li-Cor 1800
spectroradiometer (Lincoln, NE). Natural lighting conditions were sunny and
greenhouse supplemental lighting was on. After four weeks under experimental
conditions, plants were destructively harvested, dried, and treatment dry weight was
recorded.
105 5.3 Results and Discussion
5.3.1 Chemical Growth Regulation of Cuphea schumannii and Cuphea pulchra
5.3.1.1 Growth Regulation by Inhibitors of Gibberellin Synthesis
Although the general actions of the active growth retardant chemicals daminozide, chlormequat, and paclobutrazol in B-Nine®, Cycocel®, and Bonzi®
respectively are similar in the inhibition of gibberellin synthesis, plant sensitivity is
distinguishable. The results of the chemical growth retardant studies are separated by
species and further separated by growth regulator for comparison against the control.
Cuphea schumannii
C. schumannii is by nature a “leggy” plant with indeterminate growth. As noted
in previous chapters, this species possesses little potential as a successful new
introduction into the industry. However, because its growth habit is so different from C.
pulchra it was beneficial to have it as a comparative species.
In general, Cycocel® at any concentration tested did not significantly reduce
internode elongation or overall height of the plant (Table 5.3). Interestingly, only the
lowest concentration of Cycocel® at 800 ppm resulted in a statistically significant
reduction in average internode length and change in height over the controls. No other
significant difference existed between Cycocel® treated plants and control.
All concentrations of B-Nine® (2500, 3750, and 5000 ppm daminozide) produced
plants with a significant reduction in height and diameter and significantly shorter
internodes that that of control plants (Table 5.4). The change in height over the five week
106
Average Plant Average Lateral Change in Diameter # Nodes per Internode # Lateral Branch # Expanded Leaf Area † Treatment Height (cm) (cm) Main Stem Length (cm) Branches Length (cm) Leaves (cm2) Cycocel 800 ppm 23.0 ± 3.5 * 23.5 ± 1.3 16.0 ± 1.0 2.0 ± 0.3 * 4.2 ± 1.0 8.7 ± 1.4 50.4 ± 8.6 574.2 ± 78.3
Cycocel 1250 ppm 31.5 ± 2.9 24.1 ± 1.3 16.0 ± 0.4 2.6 ± 0.2 5.2 ± 0.5 8.0 ± 0.8 54.6 ± 3.0 540.5 ± 52.5 Cycocel 2000 ppm 29.4 ± 2.3 21.9 ± 2.2 15.6 ± 1.0 2.4 ± 0.2 4.0 ± 0.6 6.3 ± 1.2 43.8 ± 4.4 435.3 ± 84.9
107 Control 39.8 ± 2.9 27.5 ± 1.9 15.7 ± 0.7 3.1 ± 0.3 4.3 ± 0.7 8.3 ± 1.3 48.9 ± 4.7 629.9 ± 61.1 † Main stem is that measured to calculated change in height * Indicates treatment significantly different from control at P<0.05
Table 5.3. Effect of Cycocel® concentrations of 800, 1250, and 2000 ppm on Cuphea schumannii plant growth parameters. Values are the mean of five plants ± standard error.
Average Plant Average Lateral Change in Diameter # Nodes per Internode # Lateral Branch # Expanded Leaf Area † Treatment Height (cm) (cm) Main Stem Length (cm) Branches Length Leaves (cm2) B-Nine 2500 ppm 20.4 ± 1.8* 18.0 ± 1.1* 17.4 ± 0.9 1.7 ± 0.1* 4.0 ± 0.6 7.8 ± 0.5 49.0 ± 5.6 696.7 ± 94.4
B-Nine 3750 ppm 20.6 ± 4.1* 15.1 ± 2.4* 15.0 ± 0.6 2.0 ± 0.2* 4.2 ± 1.0 4.5 ± 0.6 39.2 ± 3.2 510.3 ± 115.4
B-Nine 5000 ppm 13.4 ± 2.8* 14.8 ± 1.6* 16.0 ± 0.4 1.3 ± 0.2* 3.6 ± 0.5 7.2 ± 0.5 45.0 ± 4.6 554.0 ± 33.8 Control 39.8 ± 2.9 27.5 ± 1.9 15.7 ± 0.7 3.1 ± 0.3 4.3 ± 0.7 8.3 ± 1.3 48.9 ± 4.7 629.9 ± 61.1 † Main stem is that measured to calculate change in height 108 * Indicates treatment significantly different from control at P<0.05
Table 5.4. Effect of B-Nine® concentrations of 2500, 3750, and 5000 ppm on Cuphea schumannii growth parameters. Values are the mean of five plants ± standard error.
period was reduced 49%, 48%, and 66% for plants treated with 2500 ppm, 2750 ppm,
and 5000 ppm respectively in comparison to the controls. Internode length was reduced
to 45%, 36%, and 58% that of the control for 2500 ppm, 2750 ppm, and 5000 ppm
respectively. Treatment with B-Nine® did not have a significant effect on any other plant
parameter measured.
Concentrations of 10 ppm, 25 ppm, and 40 ppm Bonzi® produced plants with a
significantly lower change in height, reduced diameter and reduced internode length
(Table 5.5). Change in height was reduced to 42%, 49%, and 74% that of the control for
10, 25, and 40 ppm respectively. Internode length decreased 32%, 39%, and 55% of the
control for 10, 25, and 40 ppm respectively. Treatment with Bonzi® produced overly stunted plants at the 40ppm concentration (Figure 5.2). Leaves of Bonzi® treated plants
were a darker green with smaller leaves than those of the control plants or those treated
with Cycocel® or B-Nine®.
By including a treatment group that was manually pinched at the outset of the
study, initial comparisons could be made regarding the effectiveness of using a cultural
practice, such as pinching, as a method of height control over chemical methods. Results
indicate that pinching has a significant effect on plant growth of C. schumannii (Table
5.6). Pinching significantly reduced the change in height, number of nodes, average
internode length to 54%, 25%, and 26% respectively of the control. Average lateral
branch length, however, was significantly longer in pinched plants. C. schumannii
exhibits significant apical dominance and when the apex is removed, lateral branching
increases. This produced plants with a balanced appearance in comparison to the control,
which tended to grow unidirectionally (Figure 5.3).
109
Average Plant Average Lateral Change in Diameter # Nodes per Internode # Lateral Branch # Expanded Leaf Area † Treatment Height (cm) (cm) Main Stem Length (cm) Branches Length (cm) Leaves (cm2) Bonzi 10 ppm 22.9 ± 5.4* 18.3 ± 2.1* 15.8 ± 0.7 2.1 ± 0.2* 4.4 ± 0.5 6.5 ± 0.1 45.8 ± 3.7 391.4 ± 14.1
110 Bonzi 25 ppm 20.2 ± 3.5* 15.0 ± 1.2* 15.8 ± 0.6 1.9 ± 0.3* 5.4 ± 0.9 4.8 ± 0.6 42.2 ± 3.0 441.0 ± 58.6
Bonzi 40 ppm 10.5 ± 1.8* 8.5 ± 1.9* 14.4 ± 0.8 1.4 ± 0.1* 2.4 ± 0.5 3.8 ± 1.3* 36.0 ± 3.0 229.2 ± 105.2*
Control 39.8 ± 2.9 27.5 ± 1.9 15.7 ± 0.7 3.1 ± 0.3 4.3 ± 0.7 8.3 ± 1.3 48.9 ± 4.7 629.9 ± 61.1 † Main stem is that measured to calculate change in height * Indicates treatment significantly different from control at P<0.05
Table 5.5. Effect of Bonzi® concentrations 10, 25, and 24 ppm on Cuphea schumannii plant growth parameters. Values are the mean of five plants ± standard error.
Average Average Lateral # Nodes Change in Plant Internode Branch # per Main Height Diameter Length # Lateral Length Expanded Leaf Area † Treatment (cm) (cm) Stem (cm) Branches (cm) Leaves (cm2) Pinch 18.4 ± 1.6* 24.6 ± 1.8 11.7 ± 0.6* 2.3 ± 0.2* 3.6 ± 0.3 15.3 ± 1.3* 53.7 ± 2.4 720.5 ± 48.7
Control 39.8 ± 2.9 27.5 ± 1.9 15.7 ± 0.7 3.1 ± 0.3 4.3 ± 0.7 8.3 ± 1.3 48.9 ± 4.7 629.9 ± 61.1
111 † Main stem is that measured to calculate change in height * Indicates treatment significantly different from control at P<0.05
Table 5.6. Effect of apex removal (pinch) on Cuphea schumannii plant growth parameters. Values are the mean of five plants ± standard error.
Figure 5.2. Cuphea schumannii treated with Bonzi® (paclobutrazol) plant growth regulator. The plant on the far left is the control. Continuing from left to right, plants were treated with 10, 25, and 40ppm respectively.
Figure 5.3. Pinched C. schumannii (right) vs. control (left).
112 Numerical values and statistics will reveal significant differences between
treatment groups and control, however, these values give poor indication as to the
appearance or aesthetic quality of the plant. A chemical growth retardant may be
successful at reducing internode elongation and plant height, however, if a plant is overly stunted or has an adverse aesthetic response to the chemical used, the plant is not marketable and the chemical growth retardant is unsuccessful. The purpose of conducting the evaluations was to gain “consumer” feedback as to what could be considered marketable plants. Table 5.7 summarizes the evaluation of C.
schumannii. Based only on the numerical evaluations of foliage color, vegetative
vigor, and elongation control, two treatment groups were rated as highest for plant
quality, B-Nine® 2500 ppm and B-Nine® 5000 ppm. Evaluators were also asked
to indicate the one treatment group that they believed to be the best quality plants.
Thirty percent of evaluators chose B-Nine® 5000 ppm as having the best quality
plants (Figure 5.4). Plants maintained a uniform, compact growth habit and
showed no phytotoxic or other adverse effects of the chemical.
113 Ranked "Best" Vegetative Elongation Overall Foliage color Vigor Control Treatment B-Nine 2500 4 4 3 7.6% B-Nine 3750 4 3 2 7.6% B-Nine 5000 4 4 3 30.8% Cycocel 800 3 4 3 23.1% Cycocel 1250 3 3 4 23.1% Cycocel 2000 3 3 3 0.0% Bonzi 104330.0% Bonzi 254320.0% Bonzi 403210.0% Pinch 3 4 4 7.6% Control 4 4 5 0.0%
Table 5.7. Evaluation summary for PGR treated Cuphea schumannii. Foliage color and vegetative vigor were rated on a scale of 1 to 5 (1=poor, 5= excellent). Plant elongation control was also rated on a scale of 1 to 5 (1=excessively stunted, 3= good control, 5= excessively leggy). Evaluators were also asked to indicate the treatment groups they believed to have the best quality plants. Based on numerical values and evaluator ranking, B-Nine® 5000 (indicated in bold) was the most effective PGR in controlling growth while maintaining an attractive plant.
114
Figure 5.4. Cuphea schumannii treated with 5000 ppm daminozide (B-Nine®) was chosen by evaluators to have the best overall appearance when compared with untreated controls, pinched plants, and those that had been treated with other concentrations of commercially available CGRs.
Cuphea pulchra
Cuphea pulchra “B” was the line used for this PGR study. This line is
inherently compact but maintains a bit more free form than a rounded shape like
that of line “A”. Although the species may not require a great deal of height
control, the use of PGRs may help to encourage stronger lateral branching that
would allow the plants to better withstand handling and transplanting into the
garden or landscape. Because C. pulchra is naturally a slower grower than C.
schumannii and because the outdoor air temperature and natural light conditions
were lower than during the C. schumannii trial the study was extended to 8 weeks.
115 Cycocel® concentrations of 1250 ppm and 2000 ppm significantly reduced change in height, plant diameter, internode length, lateral branch length, leaf area, plant dry weight in comparison to control plants (Table 5.8). Change in height over the eight week study period was reduced 42% and 47% from the control for
1250 ppm and 2000 ppm respectively. Internode length was reduced 27% for
1250 ppm treated plants and 31% for 2000 ppm treated plants. For C. pulchra,
significantly reducing the length of lateral branching would not be considered an
aesthetic benefit. The open lateral branching of the species gives it its appealing
shape and by shortening the lateral branches this form is lost and overly compact
(Figure 5.5).
Figure 5.5. Cuphea pulchra treated with 800, 1250, and 2000 ppm (left to right) of Cycocel® PGR. Height and internode length were significantly reduced when treated with 1250 or 2000ppm Cycocel®. However, the significant reduction in lateral branch lengths for all Cycocel® treated plants reduces the quality of the plants by reducing the attractive open branching form of the species. 116
Average Average Lateral Change in Plant # Nodes Internode Branch Height Diameter per Main Length # Lateral Length # Expanded Leaf Area Dry Treatment (cm) (cm) Stem † (cm) Branches (cm) Leaves (cm2) Wei ght (g) Cycocel 800 ppm 22.2 ± 0.9 15.3 ± 1.7 12.8 ± 0.9 2.3 ± 0.1 7.8 ± 0.4 4.2 ± 0.3* 62.6 ± 3.5 297.2 ± 27.3 2.3 ± 0.8
Cycocel 1250 ppm 16.0 ± 1.1* 11.3 ± 1.2* 13.4 ± 0.4 1.9 ± 0.2* 6.4 ± 0.7 4.2 ± 0.3* 53.2 ± 4.5* 219.7 ± 29.2* 1.7 ± 0.9* 117 Cycocel 2000 ppm 14.7 ± 2.0* 13.1 ± 0.2* 13.0 ± 0.5 1.8 ± 0.1* 6.6 ± 0.7 5.5 ± 0.9* 67.2 ± 8.5 257.1 ± 24.5* 2.0 ± 0.5*
Control 27.7 ± 1.9 21.0 ± 1.9 13.8 ± 0.5 2.6 ± 0.1 8.6 ± 1.0 8.2 ± 0.5 87.0 ± 7.3 448.8 ± 62.9 3.3 ± 0.4
† Main stem is that measure to calculate change in height * Indicates treatment significantly different from control at P<0.05
Table 5.8. Effect of Cycocel® concentration 800, 1250, and 2000 ppm on Cuphea pulchra plant growth parameters. Values are the mean of five plants ± standard error.
All three concentrations of B-Nine® resulted in plants with significantly reduced change in height, plant diameter, and average lateral branch length (Table 5.9). However, there was no significant difference in average internode elongation. Change in height was reduced 41%, 47%, and 42% from the control for 2500 ppm, 3750 ppm, and 5000 ppm, respectively. B-Nine® had no significant effect on other parameters measured.
Change in height, plant diameter, internode length, lateral branch length, and number of expanded leaves were significantly reduced for all Bonzi® treated plants, regardless of the concentration when compared with the control (Table 5.10). Change in height was reduced 60%, 62%, and 69% in relation to control for 10, 25, and 40 ppm, respectively. Internode length was reduced 38%, 35% and 38% in relation to control for
10, 25, and 40 ppm, respectively. Lateral branch length was reduced in treated plants producing plants with an overly compact appearance (Figure 5.6). Despite the reduction in height and internode length, paclobutrazol is not a good candidate for the chemical control of C. pulchra due to the high sensitivity of the species to even low concentrations.
Regardless of the concentration used, foliage of treated plants took on a crinkled appearance and were smaller and a much darker green than the controls. These features in conjunction with the stunting of lateral branching produced low quality plants.
A manual pinch of C. pulchra at the initiation of the study produced plants with a significantly reduced change in height, number of nodes, and number of lateral branches in comparison to controls after eight weeks (Table 5.11). Change in height was reduced
26% from that of the controls. The average lateral branch length of pinched plants was
118
Average Average # Nodes Change in Plant Internode Lateral per Main Height Diameter Length # Lateral Branch # Expanded Leaf Area Dry † Treatment (cm) (cm) Stem (cm) Branches Length Leaves (cm2) Wei ght (g) B-Nine 2500 ppm 16.4 ± 0.8* 12.7 ± 1.1* 14.0 ± 1.5 1.9 ± 0.2 6.6 ± 0.7 4.9 ± 0.4* 59.0 ± 5.0* 296.6 ± 52.4 1.9 ± 0.3 B-Nine 3750 ppm 14.8 ± 1.5* 12.3 ± 1.8* 12.4 ± 0.9 1.9 ± 0.2 8.6 ± 0.7 5.3 ± 0.4* 68.4 ± 8.2 395.0 ± 102.5 2.5 ± 0.6 119 B-Nine 5000 ppm 16.2 ± 1.7* 13.1 ± 1.0* 12.0 ± 0.7 2.1 ± 0.2 7.8 ± 0.9 5.4 ± 0.3* 60.6 ± 5.0 286.6 ± 7.4 2.4 ± 0.5
Control 27.7 ± 1.9 21.0 ± 1.9 13.8 ± 0.5 2.6 ± 0.1 8.6 ± 1.0 8.2 ± 0.5 87.0 ± 7.3 448.8 ± 62.9 3.3 ± 0.4 † Main stem is that measured to calculate change in height * Indicates treatment significantly different from control at P<0.05
Table 5.9. Effect of B-Nine® concentrations 2500, 3750, and 5000 ppm on Cuphea pulchra plant growth parameters. Values are the mean of five plants ± standard error.
Average Average # Nodes Change in Plant Internode Lateral Dry per Main Height Diameter Length # Lateral Branch # Expanded Leaf Area Wei ght † Treatment (cm) (cm) Stem (cm) Branches Length Leaves (cm2) (g) Bonzi 10 ppm 11.2 ± 1.6* 9.8 ± 0.6* 12.2 ± 0.5 1.6 ± 0.1* 7.4 ± 0.5 4.6 ± 0.4* 61.8 ± 6.6* 247.8 ± 60.1 2.0 ± 0.5
Bonzi 25 ppm 10.4 ± 0.5* 8.4 ± 0.6* 11.6 ± 0.5* 1.7 ± 0.0* 6.6 ± 0.5 4.0 ± 0.4* 56.4 ± 4.6* 201.3 ± 20.4* 1.8 ± 0.2* 120 Bonzi 40 ppm 8.7 ± 0.6* 6.4 ± 0.9* 10.4 ± 0.5* 1.6 ± 0.1* 3.2 ± 0.7* 3.7 ± 0.8* 39.8 ± 4.4* 141.5 ± 24.9* 1.3 ± 0.2*
Control 27.7 ± 1.9 21.0 ± 1.9 13.8 ± 0.5 2.6 ± 0.1 8.6 ± 1.0 8.2 ± 0.5 87.0 ± 7.3 448.8 ± 62.9 3.3 ± 0.4 † Main stem is that measured to calculate change in height * Indicates treatment significantly different from control at P<0.05
Table 5.10. Effect of Bonzi® concentrations 10, 25, and 40 ppm on Cuphea pulchra plant growth parameters. Values are the mean of five plants ± standard error.
Average Average # Nodes Change in Plant Internode Lateral # per Main Height Diameter Length # Lateral Branch Expanded Leaf Area Dry † Treatment (cm) (cm) Stem (cm) Branches Length Leaves (cm2) Weight (g) Pinch 20.5 ± 1.2* 23.0 ± 2.5 11.8 ± 0.2* 2.4 ± 0.1 5.4 ± 0.6* 16.5 ± 0.9* 75.8 ± 5.3 443.3 ± 19.8 3.2 ± 0.2
Control 27.7 ± 1.9 21.0 ± 1.9 13.8 ± 0.5 2.6 ± 0.1 8.6 ± 1.0 8.2 ± 0.5 87.0 ± 7.3 448.8 ± 62.9 3.3 ± 0.4 121 † Main stem is that measured to calculate change in height * Indicates treatment significantly different from control at P<0.05
Table 5.11. Effect of apex removal (pinch) on Cuphea pulchra plant growth parameters. Values are the mean of five plants ± standard error.
nearly 52% longer than controls, suggesting that removal of the apex at the outset of the
study was successful in promoting lateral branch formation. It is probable that a manual
pinch in coordination with a low concentration PGR may produce a plant shorter in
stature but with lateral branch formation to maintain in natural attractive form.
Figure 5.6. Cuphea pulchra treated with 10, 25, and 40ppm (groups left to right) Bonzi®. Although treatment with Bonzi® (paclobutrazol) significantly reduced height and internode length, the side effects of over- stunted lateral branches and unattractive leaf morphology makes Bonzi® a poor candidate as a PGR for the species.
As indicated previously, statistical values will elucidate a significant difference in
the plant produced by a particular treatment. However, these values do not provide an
indication of the aesthetics of the plants. The results of the evaluation of PGR treated C. 122 pulchra are given in Table 5.12. Based on the numerical rankings for foliage color, vegetative vigor, and elongation control, evaluators scored B-Nine® 3750ppm and
Cycocel® 800ppm equally high with a 4 for vegetative color, 4 for vigor, and a 3 for good elongation control. Evaluators were asked to indicate the one treatment that they believed to produce the best quality plants. Pinched plants received scores of 4 for
Ranked "Best" Foliage Vegetative Elongation Overall color Vigor Control Treatment B-Nine 2500 3 3 3 14% B-Nine 3750 4 4 3 29% B-Nine 5000 3 3 3 14% Cycocel 800 4 4 3 0% Cycocel 1250 3 2 3 0% Cycocel 2000 3 2 3 0% Bonzi 10 4 3 2 0% Bonzi 25 4 3 2 0% Bonzi 40 4 3 1 0% Pinch 4 4 4 43% Control 4 4 4 0%
Table 5.12. Evaluation summary for PGR treated Cuphea pulchra. Foliage color and vegetative vigor were rated on a scale of 1 to 5 (1=poor, 5= excellent). Plant elongation control was also rated on a scale of 1 to 5 (1=excessively stunted, 3= good control, 5= excessively leggy). Evaluators were also asked to indicate the treatment groups they believed to have the best quality plants. Based on numerical values and evaluator ranking, pinched plants (indicated in bold) was the most effective method for controlling growth while maintaining an attractive plant.
123 vegetative color, 4 for vigor, and 4 for moderately “leggy” and were chosen most
frequently (43% of evaluators) as being the best quality plants (Figure 5.7). B-Nine®
3750 was selected as the best overall treatment by 29% of evaluators and Cycocel® 800 was never selected as best treatment.
Figure 5.7. Control (left) vs. pinched (right) Cuphea pulchra. Although difference between the control and pinched plants are subtle, pinched plants had a significantly reduced change in height over the eight week study when compared with the control plants. Pinched plants were evaluated against nine PGR treatments and control and were ranked as the treatment group with the highest quality plants.
5.3.1.2 Growth Regulation by Ethylene-Releasing Chemical Florel®
An additional consideration surrounding propagation of C. pulchra, particularly
during the summer months, was that the species flowered continuously and provided a
small window of opportunity to take truly vegetative cuttings. It was suggested that the 124 ethylene-releasing action of ethephon, which is the active ingredient in the commercially available PGR Florel®, would aid in the prevention flowering and maintenance of vegetative stock plants. A small, preliminary trial was conducted in which mature C. pulchra plants were spray treated with a 300 ppm or 500 ppm of Florel®. Two plants were treated for each concentration. Within two weeks, all four treated plants exhibited significant defoliation, suggesting high sensitivity of the species to ethylene. Given this negative response of C. pulchra to treatment with ethephon, no further evaluation of ethylene releasing chemicals was conducted.
5.3.2 Non-Chemical Growth Regulation of Cuphea pulchra and Cuphea schumannii by
Photoselective Filters.
In light of the rising environmental concerns surrounding use of horticultural chemicals, it is important that non-chemical methods of growth regulation be explored particularly for new crop species being researched. The non-chemical method chosen for this study was control of plant growth by the manipulation of spectral composition through use of far-red selective plastic films. The film used in this study, produced by
Mitsui Chemicals, Inc. (Japan), effectively intercepts far-red wavelengths with a maximum interception at 780 or 730nm (Rajapakse et al., 1999). Although these filters are not yet available for commercial production (Rajapakse et al., 1999; M. McMahon, personal communication), continuing trials with new species will be beneficial when such materials are available in the near future to reduce costs and health risks associated with use of chemical growth regulators.
125 Two trials to evaluate the effectiveness of far-red absorbing films to reduce height
and internode elongation of Cuphea pulchra and Cuphea schumannii were conducted in
2002. Results of the two trials have been combined for both species.
The FR-absorbing filters slightly affected the R: FR ratio (Table 5.2). The readings taken mid-morning on a sunny day with the greenhouse supplemental lighting on showed that the R: FR was 1.7, 1.2, and 1.3 under FR-filter, under clear plastic, and open bench respectively. No significant differences existed between C. pulchra plants grown under the far-red filters, clear filter, or open bench conditions (Table 5.13).
Average Change in Internode # Lateral # Expanded Treatment Height (cm) # of Nodes Length (cm) Branches Leaves Far Red Filter 17.5 ± 3.1 8.4 ± 0.7 2.4 ± 0.2 7.4 ± 1.6 46.6 ± 9.8
Clear Filter 16.4 ± 4.5 8.25 ± 1.7 2.3 ± 0.4 6.3 ± 1.9 39.4 ± 11.8
Open Bench 14.7 ± 3.7 7.9 ± 1.2 2.1 ± 0.2 7.1 ± 2.1 38.0 ± 9.8 No significant difference between treatments (P<0.05)
Table 5.13. Comparative results of Cuphea pulchra plants grown under a far-red reducing filer, clear filter, or open bench (no filter) conditions. No significant differences exist between the treatments for parameters measured.
126 Use of far-red filters was also ineffective in reducing height and internode length
of C. schumannii when compared to plants maintained under non-manipulated open bench conditions (Table 5.14). C. schumannii maintained under far-red filters did have a
lower average change in height and shorter internode length than plants under open bench
conditions. The far-red filter and open bench treatments were significantly different in
the number of expanded leaves after four weeks. C. schumannii grown under the far-red
filter had significantly fewer leaves than open bench plants. The primary cause for this
is believed to be the intumescence formation on the leaves of plants maintained under the
far-red and clear filters. Incidence of intumescence became so severe under these
conditions that older, lower leaves were senescing from the plant. Although relative
humidity under the filter was not monitored, it is possible that an increased humidity
level under the plastic filters was a major contributor to the severity of intumescence
injury.
Average Change in Internode # Lateral # Expanded Treatment Height (cm) # of Nodes Length (cm) Branches Leaves Far Red Filter 30.8 ± 2.1 a 11.4 ± 0.5 a 3.2 ± 0.2 a 3.9 ± 0.7 a 17.4 ± 1.7 a
Clear Filter 33.9 ± 1.1 a 10.4 ± 0.4 a 3.8 ± 0.1 b 4.0 ± 0.5 a 20.0 ± 2.4 ab
Open Bench 33.7 ± 1.4 a 10.6 ± 0.5 a 3.7 ± 0.1 ab 4.5 ± 0.6 a 30.4 ± 4.8 b a,b Values followed by the same letters are not significantly different at P<0.05
Table 5.14. Comparative results of Cuphea schumannii plants grown under far-red reducing filter, clear filter, or open bench (no filter) conditions. 127 The concentration of pigment in the photoselective filters also reduces the quantity of light transmission, which gives the filters a shading quality. For most flowering greenhouse crops increased light quantity directly correlates with increased plant productivity. In general, the greater the light intensity, the greater the rate of photosynthesis, and the more energy available for normal plant growth and development
(Frankland, 1986). Based on the cumulative photosynthetic photon flux values of 0.08 and 0.11 mol m-2 under FR-filter and open bench conditions, respectively in April and
0.11 and 0.13 mol m-2, respectively in July, there is an observable decrease in light intensity under FR-filter conditions. This reduction in light quality may help to explain the reduction in quality of C. schumannii plants grown under FR- absorbing filters.
However, based on the increase in plant height, number of nodes, internode length, lateral branches and expanded leaves of C. pulchra plants grown under FR-filters, the decrease in light intensity did not significantly affect plant vigor of this species
5.4. Conclusions
Height control and compactness are important for production systems to maximize greenhouse space available for production and to produce plants that are healthy and compact and better able to withstand handling and transplanting. This study evaluating methods of growth regulation was necessary to provide information on the successful production of two species for which this information was previously unavailable.
The “leggy” and indeterminate growth habit of C. schumannii is the main characteristic restricting the species potential for introduction. However, this study
128 showed that chemical growth regulation provides an option to restrict this wild characteristic. The commercially available PGR B-Nine® was successful in uniformly producing healthy, compact C. schumannii specimens at a concentration of 5000 ppm. C. pulchra was responsive to PGR but lateral branching formation and elongation appeared overly sensitive to PGRs even at low concentrations. According to evaluators, C. pulchra that had been manually pinched and not treated with PGRs were significantly controlled and maintained a superior appearance over chemically treated plants.
The possible health risks associated with chemical growth regulators combined with increasing environmental awareness justifies the study of non-chemical alternatives for production of healthy and compact plants. Although use of photoselective filters has proven to be a successful method of growth regulation for some greenhouse crops, based on this study C. pulchra and C. schumannii are not responsive to the manipulation of light quality by FR-absorbing filters. Future studies evaluating the effectiveness of other non-chemical methods of growth regulation, temperature manipulation in particular, are necessary. Results of the CGR study indicate that employing the cultural procedure of pinching can be a successful non-chemical method for controlling growth of C. pulchra.
A larger scale study is necessary to determine the most effective pinching schedule to produce high quality and uniform plants. The species responsiveness to a non-chemical method of regulation is an additional characteristic that makes this species a fine candidate for introduction.
129 CHAPTER 6
PHOTOPERIODIC EFFECTS ON FLOWERING AND GROWTH
6.1. Introduction
Plant flowering is of greatest importance in plant development because it directly
influences reproductive effectiveness. Although many endogenous and external factors
are working simultaneously to regulate when a plant will flower, one of the most critical
factors is day length, or the light period.
From the standpoint of growing and producing floriculture crops, plant flowering
directly influences the salability of the plant. Being aware of the photoperiodic
requirements of a species for flowering is essential to developing a production plan and
an overall cropping schedule. The production plan of a particular species may include
mechanisms to control day length. Depending on the photoperiodic classification of the
plant as a long or short day plant, extending or reducing the light duration will influence how quickly a plant will move from a vegetative to a reproductive development phase.
One such photoperiodic mechanism, which is particularly effective in promoting the flowering of long day plants, is a day length extension or day continuation by which the
natural light period is extended with artificial lighting. Incandescent lamps work best for
130 extending the light period (or reducing the night period) because they are inexpensive and produce a sufficient amount of red light required to induce a photoperiodic response
(Nelson, 1998).
Assumptions can be made about the optimal production photoperiod of a species based on the natural day length conditions under which they flower most readily in their native habitats. Under natural conditions in areas of Oaxaca, Mexico, C. schumannii flowers from late November through July (S.A. Graham, personal correspondence) when the amount of natural daylight ranges from 11 hours (shortest days) to just over 13 hours
(longest days). C. pulchra, native to areas of Bahia, Brazil, flowers year round but most abundantly in January, February, March, June, July, August, September, October,
November, and December. Plants are exposed to a natural day length during the year of
11 to 13 hours.
One of the primary observations made during the initial greenhouse observation period was that C. pulchra and C. schumannii flowered continually when maintained under a 16 hour photoperiod in the greenhouse. Due to the long, artificially maintained day length, it was not feasible to make conclusions about species photoperiodic flowering requirements. Therefore, the objective of the following study was to begin the evaluation of photoperiodic requirements for flowering in Cuphea schumannii and
Cuphea pulchra. Specifically, the study set out to: 1) determine whether or not C. pulchra and C. schumannii have a photoperiodic requirement for flowering; and 2) define the photoperiodic requirement as obligate or facultative.
131 6.2. Materials and Methods
Plant Culture and Experimental Design
On November 18, 2003, the height, number of nodes, and number of expanded leaves of sixteen C. pulchra and sixteen C. schumannii five-week-old vegetative cuttings
individually contained in 15.24 cm diameter pots was recorded. Eight plant replicates of
both species were randomly selected and placed in one of two growth chambers. One
growth chamber was set to maintain an 8 hour photoperiod (short day, SD) with
fluorescent and incandescent lighting at 21ºC. The second chamber was set to maintain
an 8 hour photoperiod with fluorescent and incandescent light plus an 8 hour extension
on incandescent light only (long day, LD). Temperature was held at 21ºC. Eight 40W
fluorescent bulbs and ten 60W incandescent bulbs were used for both chambers. The
R:FR was measured for incandescent and fluorescent lighting together and incandescent
lighting alone. Light intensity, temperature, and relative humidity were monitored in
both chambers using appropriate sensors and data loggers (Spectrum Technologies,
Plainfield, IL) (Table 6.1). Plants were watered by hand as needed and fertilized once
per week with 200 mg N L-1 of 20-10-20 Peters complete fertilizer (Scotts Co.,
Marysville, OH). Each pot was set in a plastic saucer to contain draining water.
132
Average Average Photoperiod Average hourly Average daily Temperature Relative Treatment PPF (µmol m-2) PPF (µmol m-2) (ºC) Humidity (%)
SD 206.2 1795.6 21.1 40.6 LD 178.8 2529.8 21.7 44.8
Table 6.1. Average hourly photosynthetic photon flux (PPF), average daily PPF, average temperature (ºC) and average relative humidity (%) of the growth chambers maintaining an 8 hr (SD) or 8 hr + 8 hr extension (LD) photoperiod. Average hourly and daily PPF is calculated for the light period only.
Cuphea schumannii
Counts of visible flower buds and mature flowers (mouth of floral tube open) were conducted weekly for all C. schumannii plants. At seven weeks after initiation, C. schumannii were removed from their respective treatment conditions. Plant height, number of nodes, number of expanded leaves, number and lengths of lateral branches
(over 2 cm), number of flower buds, and number of mature flowers were recorded.
Change in plant height and average internode length was calculated. All plants were destructively harvested and leaf area and plant dry weight were determined. Digital images were taken to compare visible differences between treatments.
One-way ANOVA was used to analyze treatment effect on bud and flower formation at 3, 5, and 7 weeks, change in plant height, final number of nodes, final average internode length, final number of expanded leaves, final number of lateral branches, leaf area, and dry weight. If the P value produced by ANOVA was <0.05, a
133 Tukeys pairwise multiple comparisons test was performed. Sigma Stat statistical software was used to analyze the data (Jandel Corp. / SPSS, Chicago, IL).
Cuphea pulchra
Counts of visible flower buds and mature flowers were conducted weekly for all
C. pulchra plants. At eleven weeks after experiment initiation, only C. pulchra held under the LD photoperiod were removed from the treatment conditions. C. pulchra plants under the 8 hour photoperiod remained under treatment conditions. Plant height, number of nodes, number of expanded leaves, number and lengths of lateral branches
(over 2 cm), number of flower buds, and number of mature flowers were recorded.
Change in plant height and average internode length was calculated. Plants were destructively harvested to determine leaf area and plant dry weight. Digital images were taken to compare development of C. pulchra held under the two treatment conditions.
One-way ANOVA was used to analyze treatment effect on bud and flower formation of
C. pulchra maintained under an 8 hour or an 8 hour + 8 hour extension photoperiod at 3,
5, 7, 9, and 11 weeks after initiation of the experiment.
At 13 weeks after initiation, five of the eight C. pulchra remaining under the SD photoperiod were transferred to the 8 hour+ 8 hour extension photoperiod conditions.
Plant height, number of nodes, and number of lateral branches were recorded for the five plants being transferred. The purpose of this transfer was to determine the duration of time required for C. pulchra held under short days to visibly enter its reproductive development phase once it is under a LD photoperiod. Visible flower bud and mature flower counts were conducted weekly for the five transferred plants and the three plants
134 remaining under the 8 hour photoperiod. At six weeks after transfer to long days (20 weeks after experiment initiation), the five C. pulchra were removed from the treatment conditions. Plant height, number of nodes, number of expanded leaves, number and lengths of lateral branches (longer than 2 cm), number of flower buds, and number of mature flowers were recorded. Change in plant height and average internode length was calculated. Plants were destructively harvested to determine leaf area and plant dry weight. A one-way ANOVA was used to analyze treatment effect on bud and flower formation of C. pulchra maintained under SD or LD photoperiod at 1,3, and 6 weeks after transfer of plant to the 8 hour + 8 hour extension.
Visible flower bud and mature flower counts were continued weekly for the three plants remaining under the 8 hour photoperiod. At 23 weeks after initiation, the three plants were removed from the treatment condition. Plant height, number of nodes, number of expanded leaves, number and lengths of lateral branches (longer than 2 cm), number of flower buds, and number of mature flowers were recorded. Change in plant height and average internode length was calculated. Plants were destructively harvested to determine leaf area and plant dry weight.
Because plant parameter data for these different groups were not collected simultaneously, data had to be transformed to make statistical comparisons possible.
Plant parameter data was divided by the number of weeks the plant had been under experimental conditions, 11, 20, and 23 week for LD, SD transferred to LD, and SD respectively. This allowed data to be analyzed as a rate of change or increase over a one
135 week period. Data was analyzed by a one-way ANOVA. If the P value produced by the
ANOVA was <0.05 a Tukeys pairwise multiple comparisons test was performed. Sigma
Stat statistical software was used (Jandel Corp. / SPSS, Chicago, IL).
6.3. Results and Discussion
6.3.1 Cuphea schumannii
Regardless of the photoperiodic conditions, C. schumannii plants held under an 8
hour (SD) or and 8 hour+8 hour extension (LD) had visible bud set and some flowering
by the third week under the experimental conditions. The lack of difference in the timing
of visible bud set and flower would suggest that C. schumannii lacks or has a very weak
photoperiodic requirement for flowering. After week three, there was a significant
difference in the number of visible buds and mature flowers produced between the two
treatment groups (Figure 6.1). C. schumannii plants maintained under the 8 hour photoperiod had a significantly (P<0.05) higher number of visible buds and mature flowers than those plants maintained under the 8 hour extension.
Appearance of plants maintained under the two photoperiodic conditions was quite different (Figure 6.2). C. schumannii plants held under the 8 hour+8 hour extension were excessively “leggy”. Fully expanded leaves exhibited a reddish color beginning at the margins and moving inward and there was noticeable senescence of lower leaves. C. schumannii under the 8 hour photoperiod were much shorter in stature, foliage was a healthy green color, and very low incidence of lower leaf senescence. In addition to the increased exposure to FR light due to the incandescent day length extension, another
136 50 45 SD buds LD buds 40 SD flowers 35 LD flowers 30 25 20 15
Number of Buds or Flowers Flowers or Buds of Number 10 5 0 week 3 week 5 week 7
Figure 6.1. Graphical depiction of the difference in the number of visible buds and flowers produced on Cuphea schumannii under short-day (SD) and long-day (LD) photoperiods at 3, 5, and 7 weeks after experiment initiation. Points represent the mean number of buds or flowers of eight plants per treatment. Vertical bars indicate the standard errors of the mean.
137
Figure 6.2. Digital image comparing the morphological differences between C. schumannii maintained under short day (right) and long day (left) photoperiod for 7 weeks.
possible cause for this difference in appearance, particularly with regard to leaf color and
senescence, is difference in soil moisture. Plants under the 8 hour + 8 hour extension
were exposed to an addition 8 hours of light during which the plants could lose water
through transpiration. There was no significant difference in the average temperatures of
the two growth chambers (Table 6.1) to imply that temperature could also be a
contributing factor to the aesthetic differences. It is possible that the water available to
the maturing plants was not enough to facilitate 16 hours of light per day and the
observable differences in foliar health were due to water deficiency stress. This may also
be the primary cause for the decrease in the number of visible buds and mature flowers
on the LD plants. 138 There was no significant difference in the number of nodes, average internode
length, average lateral branch length, or leaf area due to photoperiodic treatment (Table
6.2). The significant differences observed between the two photoperiodic treatments can be explained by the increase in far-red light in the long-day extension. The average increase in height was significantly higher in the 8 hour + 8 hour extension plants.
Incandescent lamps are high in far-red wavelengths and fluorescent lamps high in red wavelengths (Dole and Wilkins, 1999). When R:FR measurements were taken under the combined and fluorescent and incandescent and incandescent alone, the R:FR were 1.0 and .73, respectively. This confirms that plants grown under the extended LD were exposed to eight additional hours of light high in far-red wavelengths. Light with a high
red to far-red light ratio reduces internode elongation, as observed with studies utilizing
FR-absorbing filters. Conversely, plants that are exposed to a lower R:FR will have
increased stem elongation, which is explained by the shade avoidance response.
Therefore, the significant increase in height of C. schumannii maintained under LD is
explained by the increased exposure to far-red light supplied by the incandescent light
extension. Increased far-red light increases apical dominance in plants, which results in
reduced lateral branching. This aids in explaining why plants under SD exposed to lower
amounts of FR light had significantly more lateral branches that those C. schumannii
grown under the incandescent extended LD. SD photoperiod plants had significantly
more expanded leaves. This was expected due to the senescence of lower leaves in the
LD photoperiod plants. However, SD photoperiod plants had a significantly lower dry
weight, suggesting that despite the considerable leaf loss under the LD photoperiod,
plants were able to maintain a significant amount of dry mass in the stems.
139
Average Average Lateral Change in # Nodes per Internode # Expanded Lateral Branch Leaf Area Dry Weight † 2 Photoperiod Height (cm) Main Stem Length (cm) Leaves Branches Length (cm) (cm ) (g) a a a a a a a a SD 51.0 ± 3.817.3 ± .5 3.7 ± .2 35.9 ± 2.7 4.3 ± .5 7.4 ± 2.7 721.9 ± 46.1 7.2 ± .6 140 LD 69.55 ± 3.8 b 18.9 ± .5 a 4.3 ± .2 a 25.4 ± 2.7 b 2.0 ± .5 b 10.8 ± 2.7 a 604.3 ± 46.1 a 10.7 ± .6 b † Main stem is that measured to calculate change in height a,b Values followed by the same letters are not significantly different at P<0.05
Table 6.2. Comparison of plant growth parameters of Cuphea schumannii maintained for 7 weeks under short-day (SD) or long-day (LD) photoperiodic conditions. Values are given as treatment means ± standard error (n=8).
6.3.2 Cuphea pulchra
At seven weeks after initiation of the experiment, a statistically significant difference in the number of visible buds was apparent between the photoperiodic treatments (Table 6.3). At 11 weeks, C. pulchra plants under the 8 hour + 8 hour extension averaged 133 visible buds and 13 mature flowers while plants under the 8 hour photoperiod exhibited no characteristics of entering the reproductive development phase.
This early observation suggested that C. pulchra does in fact have a photoperiodic requirement for flowering. Figure 6.3 shows the difference in plant development between the two photoperiodic treatments after 11 weeks under SD or LD photoperiod.
C. pulchra plants were transferred from the SD photoperiod to the LD photoperiod to determine the duration of time required for plants to set visible bud under an inductive LD photoperiod. Plants remaining under the SD photoperiod were observed to determine how long until and if plants would flower under the 8 hour photoperiodic treatment. At three weeks after transfer to the LD photoperiod, transferred plants had significantly more visible buds than those remaining under the 8 hour photoperiod (Table
6.4). Six weeks after being transferred to the inductive photoperiod, all C. pulchra plants had at least one mature flower and averaged 110 visible buds. Plants under the 8 hour photoperiod exhibited visible bud set at 20 weeks after experiment initiation.
141
Week 3 Week 5 Week 7 Week 9 Week 11 Vi si bl e Vi si bl e Vi si bl e Vi si bl e Vi si bl e Photoperiod buds Flowers Buds Flowers Buds Flowers Bud Flowers Bud Flowers a a a a a a a a a a SD 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 b a a a b a b a b b 142 LD 1.4 0.5 0.6 1.6 7.1 0.9 66.5 0.9 132.9 12.5 a,b Values followed by different letters are not significantly different at P<0.05
Table 6.3. Comparison of the average number of buds and flowers visible on C. pulchra plants held under short-days (SD) or long-days (LD) at 3, = 5, 7, 9, and 11 weeks after experiment initiation. Values given are treatment means (n=8).
Figure 6.3. Digital image comparing the morphological difference of C. pulchra plants that had been maintained under a long-day (left) and a short-day (right) photoperiod for 11 weeks.
Week 1 Week 3 Week 6 Vi si bl e Vi si bl e Vi si bl e Photoperiod buds Flowers Buds Flowers Buds Flowers SD 0.0 a 0.0 a 0.0 a 0.0 a 4.7 a 0.0 a a a b a b a SD → LD transfer 1.0 0.0 16.7 0.0 110.3 2.6 a,b Different superscript letters indicate statistically significant difference between treatment groups (P<0.05)
Table 6.4. Comparison of the average number of buds and flowers visible on C. pulchra plants under short-days (SD) or plants that had been transferred from short-days to long-days at 1, 3, and 6 weeks after plant transfer to LD.
At 11, 20, and 23 weeks Cuphea pulchra plants were removed from long-day, short-day transfer to long-day, and short day photoperiodic conditions respectively. 143 Because of the variability in the timing of growth parameter data collection for the C. pulchra treatment groups, data was converted to rate of change (or increase) per week in order to statistically compare treatment effects (Table 6.5). Results indicate that the weekly increase in the number of nodes, internode elongation, number of lateral branches, number of visible buds and dry weight was significantly increased in those plants that were maintained under the LD photoperiod when compared with those that were maintained under SD or transferred to LD. The increase in the number of nodes and increased internode elongation are explained by exposure to increased FR provided by the low-intensity light from the incandescent lighting used for the LD extension. As exposure to far-red light increases, stem elongation increases as a shade avoidance response (Thomas and Vince-Prue, 1997; Aphalo et al., 1999). Although statistically different, the average weekly increase in internode length of plants under SD and LD photoperiodic conditions did not vary greatly, only by 0.1 cm.
As discussed previously for C. schumannii, exposure to increased far-red light promotes apical dominance and suppresses initiation of lateral branching. However, based on results for Cuphea pulchra, plants maintained under short-days developed significantly fewer lateral branches. This is significant for two reasons. The first being that plants transferred from short-days and those maintained entirely under short-days were considerably older, nine and twelve weeks respectively, than plants maintained for
11 weeks under long-days. The difference in age alone would suggest that the SD plants would have an increased number of lateral branches. The second point of significance is
144
Average Average Lateral Change in # of Nodes Internode Branch # # Visible Photoperiodic Height per Main Length # Lateral Length Expanded Flower # Mature Leaf Area Dry † 2 Treatment (cm) Stem (cm) Branches (cm) Leaves Buds Flowers (cm ) Wei ght (g) a a a a a a a a a a LD (11 weeks) 3.6 ±1.9 0.2 ± 0.0 0.2 ± 0.0 0.9 ± 0.1 2.5 ± 0.2 18.1 ± 1.3 12.1 ± 1.6 1.1 ± 0.4 83.9 ± 4.2 1.5 ± 0.0 SD → LD (20 weeks) a b b b a b b a a b
145 3.0 ± 0.4 1.3 ± 0.1 0.1 ± 0.1 0.3 ± 0.0 1.9 ± 0.3 10.1 ± 1.1 4.7 ± 1.3 0.1 ± 0.1 66.6 ± 6.5 1.1 ± 0.1 SD (23 weeks) 2.8 ± 0.1 a 1.4 ± 0.0 b 0.1 ± 0.0 b 0.3 ± 0.1 b 1.7 ± 0.1 a 11.6 ± 1.5 ab 2.9 ± 0.6 b 0.1 ± 0.0 b 71.6 ± 11.8 a 1.0 ± 0.1 b
† Main stem is that measured to calculate change in height a,b Values followed by the same letters are not significantly different at P<0.05
Table 6.5. Plant growth parameters of Cuphea pulchra plants maintained under long-days (LD) (n=8), transferred from short-days (SD) to long days (LD) (n=5), and maintained under short-days (SD) (n=3). Data is given as change (or increase) per week ± standard error in order to statistically compare photoperiodic treatment effects.
that due to increased exposure to end-of-day far-red light, the expectation would be for plants under a LD photoperiod would have fewer lateral branches as result of FR induced apical dominance. This suggests that perhaps C. pulchra is less sensitive to this type of
FR induced response. Additional information about light intensity and quality of native habitat locations for C. pulchra is necessary before drawing any conclusions as to the inherent shade (increased FR) tolerance of the species.
C. pulchra maintained under LD photoperiod also exhibited a greater increase in the number of visible buds exhibited per week in comparison to plants maintained under
SD or those that were transferred to LD. This indicates that C. pulchra does have a photoperiodic requirement for flowering and can be classified as a facultative long-day plant. However, the increase in FR light available to these plants may also play a role in promoting bud formation. In LDP barley (Deitzer et al., 1979) and ryegrass (Taiz and
Zeiger, 1998), far-red light has been shown to promote flowering when compared with plants maintained under continuous white or red light (Taiz and Zeiger, 1998).
6.4 Conclusions
Identifying the light duration requirements for the flowering of C. schumannii and
C. pulchra is a significant step in the development of cultural information for production.
Under the artificially maintained photoperiods of 8 hour and 8 hours + 8 hour incandescent extension, C. schumannii flowers regardless of the photoperiod, classifying it as a day neutral species or a weak facultative LD plant. The 13 week delay in flowering of C. pulchra maintained under the 8 hour photoperiod in comparison to those plants maintained under a 16 hour extended photoperiod, indicates that this species does
146 have an optimal photoperiodic requirement for flowering. Based on this study C. pulchra can be tentatively labeled as a facultative long day plant. However, further evaluation of flowering of C. pulchra under intermediate photoperiods (e.g. 10, 11, 12, 13, 14 hours) will be necessary to determine the critical day length (CDL) and optimum photoperiod for controlled production of the species.
147 CHAPTER 7
ROLE OF TRICHOMES AND TRICHOME EXUDATES IN THE RESISTANCE OF
Cuphea pulchra TO GREEN PEACH APHID
7.1 Introduction
During the initial greenhouse observation period (Chapter 2), it was noted that C. pulchra displayed a deterrence to aphid settling, while other species, particularly C. schumannii, housed in the same area were experiencing significant aphid infestation.
This prompted the design of further experiments to identify the source of the deterrence.
Presence of glandular trichomes on stem, leaf, or flower tissue of Cuphea, is characteristic of many species of the genus and one, which poses an obstacle to agronomic cultivation (Hisinger and Knowles, 1984). However, this characteristic can be viewed as a benefit for those species possessing ornamental potential. In general, the antimicrobial and insect repellent properties of trichome resins make them an important component of the chemical defense mechanism of plants (Levin, 1973). A study by
Amarasinghe et al. (1991) surveying taxonomic importance of trichome diversity in
Cuphea identified seven morphological types of trichomes for 136 species studied (Table
7.1) . Resin-secreting glandular trichomes were found on 92% of the species studied; they were abundant on stems and flowers and less common on leaves.
148 Trichome Type Characteristics Location
multicellular, multiseriate, resin-secreting TYPE 1 glandular trichomes with bulbus expanded stems, flowers bases and narrow elongated necks unicellular, spine-like, thick-walled TYPE 2 tuberculate trichomes
a expanded base, obliquely protruding spines leaves
b expanded base, no spines (unique to one species)
c spine-like, no enlarged base stems, exterior floral tube
unicellular, biarmed, thick-walled, tuberculate TYPE 3 trichomes with the arms sessile and unequal leaves, stems in length one-to-four celled, uniseriate, usually thin- TYPE 4 stems walled trichomes
unicellular, elongated trichomes with TYPE 5 stamen filaments tuberculate, moderately thick walls
TYPE 6 flattened, unicelular trichomes interior floral tube
multicellular, globose, sessile, glandular rare, only found on three TYPE 7 trichomes without a neck as in type 1 species
Table 7.1. Defining characteristics and plant surface locations of the seven types (and sub-types) of trichomes on Cuphea plant surfaces as identified in Amarasinghe et al. (1991) in a survey of 136 species in the genus.
149 Phytochemical investigations of whole tissues of some species in the genus have shown that Cuphea produce fatty acids, sterols, triterpenes, tannins, and flavonoids as main secondary metabolites (Catorena et al and references cited therein, 2003; Santos et al, 1995). There is no literature reporting the identification of the active compounds secreted specifically by Cuphea glandular trichomes or the roles these compounds play in insect deterrence or resistance. However, extensive studies have been carried out in a number of other species to evaluate the influence of exudate chemicals on specific insects
(Wagner, 1991). Trichome exudate composition of members of the Solanaceae family, such as wild potato, wild tomato, and tobacco (Neal et al., 1990; Goffreda et al., 1989;
Johnson et al., 2002; Puterka et al., 2003) have been of particular interest well as ornamentals like petunia (Chortyk et al., 1996; Chortyk et al., 1997).
A class of compounds common to exudates of glandular trichomes of the above- mentioned plants is sugar esters. Sugar esters are composed of short chain fatty acids
(C2-C10) esterified to two or more of the hydroxyl groups of either glucose or sucrose
(Chortyk et al., 1997). Commercially produced sucrose esters have been used as ingredients in the food and detergent industry as emulsifiers, stabilizers, wetting agents, and surfactants. However, investigations into the role natural sugar esters derived from plant exudates in insect resistance has been a more recent endeavor (Chortyk et al. 1996;
Puterka et al., 2003).
The urgency to evaluate sugar ester producing species springs mostly from the need to develop insect-resistant cultivars to reduce the use of chemical insecticides.
Because sugar esters are already being synthetically produced for the food industry with
FDA approval, these compounds offer a safe and environmentally friendly alternative to
150 harsh chemical insecticides. Nicotiana species are a particularly large area of sugar ester
study. Both glucose and sucrose esters have been characterized from Nicotiana species
(Severson et al. 1991). When compared with activity of other leaf surface compounds in sixty-two tobacco lines, only sugar ester levels were negatively correlated with aphid resistance (Johnson et al., 2002). These results suggested to researchers that a number of the tested tobacco lines could be used for natural sugar ester biorational insecticides, or for development of aphid resistance cultivars through breeding programs.
As a result of intensive selection, cultivated tomato has lost many of the insect resistant characteristics of its wild relative, thus prompting further exploration into the role of glandular exudate compounds in insect deterrence/resistance. Goffreda et al.
(1989) found that leaf rinses of wild tomato Lycopersicon pennellii deterred settling of potato aphid. A glucose ester, localized in the glandular exudate of leaf trichomes, was determined to be the active deterrent. Results of bioassays of purified glucose esters indicated a significant positive correlation between aphid deterrence and increasing concentrations of glucose esters (Goffreda et al., 1989). Subsequent work with wild tomato utilized graft chimeras (grafts of genetically different plants to yield shoots in which one of the three primary tissues is different that the other two) to determine the role of leaf surface tissues in insect resistance (Goffreda et al, 1990). They determined that epidermal features of the leaf alone accounted for aphid deterrence and that the resistance was correlated with the presence of trichome secreted glucose esters.
Studies of wild potato (Solanum berthaultii) trichome exudate yielded similar results to those of wild tomato. Removal of trichome exudate droplets from wild potato leaflets significantly increased the fraction of green peach aphid feeding on leaflets
151 suggesting a strong inhibition of feeding by the trichome exudates (Neal et al., 1990).
Sucrose esters were identified as the predominant components of the trichome exudate and subsequent bioassays of the isolated sucrose ester showed that settling and probing of the green peach aphid were affected only when the aphids were in direct physical contact with the compound (Neal et al., 1990).
Study of petunia trichome exudates has revealed a significant role of sugar esters in resistance of sweet potato whiteflies (Kays et al., 1994). Structural investigation of these sugars disclosed that petunia trichomes secrete two glucose and four sucrose esters
(Chortyk et al., 1997). Bioassays of the individual sugar esters indicate that toxic activity could be ascribed to two of the six petunia sugar esters, which prompted a key conclusion that a specific acylation pattern and polarity of the molecule is required in the sugar ester structure to exhibit maximum whitefly toxicity (Chortyk et al., 1997; Chortyk et al.,
1996).
There is some debate surrounding the mechanism of sugar ester deterrence of aphid settling. One argument is that the viscous properties of sugar esters act to entrap these pests. However, Goffreda et al. (1989) contend that resistance cannot be completely attributed to the viscous properties because low concentrations of these compounds produce a microscopic film yet maintain significant deterring characteristics.
If aphids will not settle and feed, the probable cause of death is starvation (Neal et al.,
1990; Goffreda et al., 1989). Another argument is that hydrolysis of the ester linkage would produce free fatty acids which would act as toxic agents themselves if absorbed through the insect cuticle. Puterka et al. (2003) dispute this theory on the basis that sugar
152 ester structure does not lend itself to hydrolysis in the short time frame that it is toxic and it is doubtful that free fatty acids would be a plausible mechanism. Instead, they suggest suffocation as a probable cause of aphid mortality.
Regardless of the mechanism of resistance, some sugar esters exhibit considerable insecticidal qualities. Synthetic sucrose esters are a relatively new class of insecticidal compounds that maintain the same contact toxicity and rapid knock-down ability to soft-bodied arthropods as natural sucrose esters (Puterka et al., 2003).
Investigation of the structure-function relationship of synthesized sugar esters has shown that changes in the fatty acid and sugar components may result in variable levels of insecticidal activity as well as the range of arthropods the sugar ester is effective against
(Puterka et al., 2003; Chortyk et al., 1996). This unique chemistry allows opportunity to custom-design insecticides specific to a particular arthropod pest. Because sucrose esters are already being used as food ingredients, these compounds are especially attractive as safe and effective insecticides (Puterka et al., 2003).
The purpose of the following studies was to confirm the deterrence of green peach aphid settling on Cuphea pulchra and begin the investigation into the mechanism of resistance. Therefore, the objectives of the following three studies were to: 1) confirm initial greenhouse observation of the deterence of Cuphea pulchra to Myzus persicae
(green peach aphid) through choice and no-choice settling assays; 2) determine if C. pulchra glandular trichome exudates affect aphid settling preference; and 3) identify the active resistance compound in the glandular trichome exudate of C. pulchra.
153 7.2. Materials and Methods
7.2.1. Identification of Trichome Types
Plant surface trichomes of Cuphea schumannii and Cuphea pulchra were identified by Scanning Electron Microscopy (SEM) at the OARDC/OSU Molecular and
Cellular Imaging Center. Excised leaf, stem, and floral tissue samples of both species were placed in fixative (3% gluteraldehyde, 2% paraformaldehyde in 0.1 M potassium phosphate buffer, pH 7.4), vacuum infiltrated, and placed on a tissue rocker overnight.
Tissue samples were rinsed three times for 15 minutes in 0.1 M potassium buffer.
Samples were dehydrated by a series of rinses in EtOH: 25% for 15 minutes, 50% for 15 minutes, 75% for 15 minutes, 100% for 15 minutes. The final rinse was repeated two additional times. Tissues were then critical point dried, mounted on stubs, and sputter coated with platinum (step completed by Dave Fulton of the OARDC/OSU Molecular and Cellular Imaging Center). Samples were scanned (Hitachi S-3500N Scanning
Electron Microscope) and digital images recorded.
7.2.2. Whole Plant-Aphid Settling Assay
Plant and Aphid Culture.
Four-week old cuttings of C. schumannii and C. pulchra (line “B”) were transplanted into 15.24 cm diameter plastic pots using soilless media (Metro-Mix 360,
Scotts Co., Marysville, OH) two weeks prior to assay initiation and maintained in a growth chamber with a 16 hour photoperiod (approximately 260 µmol m-2) and
21ºC/18ºC light/dark temperature cycle. Green peach aphid (Myzus persicae) colonies
were started on two ornamental pepper plants three weeks prior to the first assay. Plants
154 were covered in double layer mesh to contain aphids and were placed in a growth chamber with a 21ºC /18ºC light/dark temperature cycle and 16 hour photoperiod.
Cage Construction
Nine 25.4 cm x 50.8 cm x 50.8 cm wood frame cages were constructed from fir stock lumber. A 55.9 cm x 30.5 cm piece of 1.3 cm plywood served as the base (Figure
7.1). Two coats of white exterior latex paint were applied to all wood surfaces. Thrips grade greenhouse screening (Hummert Intl., Earth City, MO) was attached to the sides, top, and back of the cage frame. Eight-mil clear vinyl was attached to the front of the cage frame. Weather stripping was applied to the bottom of the cages to seal against the base. Eyelet screws were attached to the top side rails to support the subirrigation apparatus of clear PVC tubing (Fisher Scientific, Pittsburgh, PA) connected to 65 mm x
35 mm x 80 mm plastic funnels (Fisher Scientific, Pittsburgh, PA) using quick disconnects (Fisher Scientific, Pittsburgh, PA). A 3 cm section of weather stripping was removed from both sides of the cage to allow the tubing into the cage where they rested in 20.3 cm planter saucers.
Experimental Design.
Three treatment groups were used in this study: choice (one C. pulchra and one
C. schumannii in a cage), no choice (two C. pulchra in a cage), and no choice (two C. schumannii in a cage). There were three replicates of each treatment. One replicate of each treatment was placed in each of three growth chambers set to maintain a 16 hour
155 photoperiod with a 21ºC /18ºC light/dark temperature cycle. A plastic Petri dish containing 8-10 adult green peach aphids was introduced into each cage. Plants were subirrigated twice weekly over the two week study period.
Evaluation and Analysis.
At initiation of the assay, height, number of expanded leaves, and number of lateral branches were recorded. A small mark was made on the stem just below the oldest set of non-expanded leaves using black permanent marker. This was to assist in determining the number of leaves that were produced and expanded in the two week time period. After two weeks, height, number of lateral branches, number of expanded leaves since initiation, number of open flowers, and total number aphids (adults and immatures) were recorded for each plant. Leaf area (Li-Cor, Lincoln, NE) of old and newly expanded leaves was determined separately and dry weight was recorded after plant tissue had dried for 96 hours in a drying oven. The assay was repeated three times.
The number of aphids settling under choice and no choice conditions was compared between species. Plant parameter data for choice and no-choice conditions was analyzed separately for each species. Data was analyzed by a one-way ANOVA. If the P value produced by the one-way ANOVA was <0.05, a Tukeys pairwise comparison test was performed. Data analysis was performed using Sigma Stat statistical software
(Jandel Corp. /SPSS, Chicago, IL).
156
Figure 7.1. Wood frame cages constructed for the whole plant aphid settling assay conducted under growth chamber conditions. Cages were constructed to facilitate the sub irrigation of two plants in 15.24 cm diameter pots.
7.2.3 Adult Green Peach Aphid Preference Assay
Cage Construction.
Four, 5 mm holes were drilled in a 9 cm diameter plastic Petri dish. A hole of the same diameter was drilled through the center of the screw-on lids of four 4.5 cm x 2.5 cm plastic liquid scintillation vials (Wheaton Scientific, Millville, NJ). The hole drilled in the center of the lid of the vials corresponded to the position of the hole in the bottom of the Petri dish. Lids were cemented in place using a PVC cement adhesive. Once adhesive was dried and lids were firmly cemented, vials were filled with water, covered with a 3 cm X 3 cm piece of Parafilm (American National Can, Neehah, WI), and
157 screwed into the attached lids. Cage “caps” to fit over the top of the Petri dish were created by stapling a 30 cm x 11 cm piece of thrips grade greenhouse screening to create an open cylinder. A 11 cm x 11 cm piece of 8 mil clear vinyl was attached to one end of the open cylinder using silicon (Figure 7.2). Twelve cages were constructed.
Figure 7.2. Cages designed for the aphid settling preference assay. One C. pulchra cutting was washed with EtOH to remove trichome exudates droplets from stems and another cutting remained untreated. Cutting stems were inserted into opposite holes in the Petri dish into the water reservoirs (left). Greenhouse screening cylinders with a clear vinyl top were used to enclose plant material and aphids (right).
158 Plant and Aphid Culture
Twenty-four fresh Cuphea pulchra (line “B”) vegetative stem cuttings taken from greenhouse stock plants were used directly in this study. Adult green peach aphids were removed from the colony stock plant (chrysanthemum) that had been held under growth chamber conditions of a 16 hour photoperiod with a 21ºC/18ºC light/dark temperature cycle. Aphids were held in a closed Petri dish and starved for four hours.
Experimental Design
Twelve C. pulchra cuttings were dipped in 75% (V:V) aqueous ethanol (EtOH) for 10 seconds, rinsed with distilled water, dipped in 75% EtOH for 10 seconds, and rinsed with distilled water. The twelve other cuttings remained untreated. All cuttings were trimmed at the base to 10 cm in length, and leaves were removed leaving only two pair of uppermost fully expanded leaves. One treated (dipped in EtOH) and one untreated cutting were inserted into opposite holes in the bottom of each Petri dish and into the water filled vial. Five adult aphids were place in the middle of each Petri dish cage. Cage “caps” were placed over the Petri dishes containing the cuttings. The twelve cage replicates were placed in a growth chamber set to maintain a 16 hour photoperiod with a 21ºC/18ºC light/dark temperature cycle. The assay was repeated one additional time.
Evaluation and Analysis.
Aphid settling was recorded 24 and 48 hours after aphids were introduced into the cages. The number of aphids (adults and immatures) on leaf or stem tissues was recorded
159 for treated and untreated cuttings. Data comparing the total number of aphids settled on
treated versus untreated cuttings as well as data comparing the number of aphids settled
on stem or leaf tissue was analyzed by one-way analysis of variance. If the P value
produced by the one-way ANOVA was <0.05, a Tukeys pairwise comparison test was
performed. Data analysis was performed using Sigma Stat statistical software (Jandel
Corp. /SPSS, Chicago, IL).
7.2.4 Analysis of Cuphea pulchra Glandular Trichome Exudates
Sample preparation
Vegetative branches of C. pulchra (line “C”) were removed from the greenhouse
stock plant. Leaves were removed and the branches were cut into 5 cm (approximately)
pieces. Each stem piece was washed in 100% EtOH for 10 seconds. Stem wash was
transferred to a 50 mL round-bottom flask and evaporated to dryness using a Rotavapor
R-114 (Büchi, Switzerland). Sample was re-suspended in 1 mL tetrahydrofuran (THF)
and a 1:10 dilution of the sample was made using THF. The standard used was sucrose
octanoate EP (40%) (Applied Power Concepts, Inc., Anaheim, CA) which is a liquid a
room temperature. One gram of standard was suspended in 1 mL 100% EtOH. A 1:10
dilution of the suspension was made using 100% EtOH.
Thin Layer Chromatography (TLC)
Samples were chromatographed on a 20 x 20 cm plate coated with a 0.1 mm
layer of silica (Eastman Kodak, Rochester, NY) using a 40:30:30 (V:V:V) of toluene,
95% EtOH, and ethyl acetate as the solvent system. Once the solvent front advanced 15
160 cm (from the bottom of the plate), the plate was removed from the developing tank and
allowed to dry by solvent evaporation in a fume hood. Sugar esters were visualized by
spraying a mixture containing 1 g of urea, 4.5 mL of 85% H3PO4, and 48 mL of water
saturated n-butanol. The plate was allowed to dry in the fume hood and was then
transferred to a 110ºC oven for 15 minutes to develop the urea-sugar complexes. The
plate was removed from the oven and allowed to cool. Retention index (distance center
of the band has moved from starting line divided by the distance the mobile phase moved
during the elution period) and band widths were recorded.
7.3 Results and Discussion
7.3.1 Identification of Trichome Types by SEM
Most plant surfaces bear trichomes. Trichomes may be simple hairs that deter
herbivores, guide pollinators, or affect photosynthesis, leaf temperature, or water loss
(Wagner, 1991). Cuphea exhibits more diversity in trichome morphology than any other genus in the family Lythraceae (Amarasinghe et al., 1991). Many species bear several
types on leaves, stems, and flowers and nearly all species display at least one type. C.
schumannii plants possess three of the seven types as described by Amarasinghe et al.
(1991). C. schumannii bear one to four celled, thin walled trichomes (TYPE 4) on the stems, unicellular elongated trichomes with moderately thick walls (TYPE 5) inside the floral tube (Figure 7.3), and unicellular, thick-walled trichomes with expanded bases and obliquely protruding spines (TYPE 2a) on stems, leaves and exterior of the floral
tube (Figure 7.4) .
161 C. pulchra possesses trichomes on stems, underside of leaves and exterior floral
tube that are unicellular, thick-walled and erect with protruding spines (TYPE 2c), TYPE
4 trichomes on the underside of leaves and interior/exterior floral tube, and multicellular
resin-secreting glandular trichomes (TYPE 1) on stems and exterior floral tube (Figure
7.5). The significant difference, with regard to trichome morphology, between these two
species is C. pulchra yielding glandular secreting trichomes (Figure 7.6).
Figure 7.3. Scanning electron microscope image of the interior floral tube surface of Cuphea schumannii. The trichomes present on the interior surface of the floral tube are TYPE 5 trichomes, which are unicellular, elongated trichomes with moderately thick walls.
162
TYPE 4
TYPE 2a
Figure 7.4. Scanning electron microscope image of the stem surface of Cuphea schumannii. Trichomes present on the stem surface are TYPE 2a trichomes, which are unicellular, thick-walled trichomes with expanded bases and obliquely protruding spines and TYPE4 trichomes, which are one to four celled and thin walled.
163 TYPE 1
TYPE 2c
Figure 7.5. Scanning electron microscope image of the exterior floral tube surface of Cuphea pulchra. Trichomes present on the exterior floral tube surface are TYPE 2c, which are unicellular, thick-walled and erect trichomes with protruding spines and TYPE 1 trichomes, which are multicellular resin-secreting glandular trichomes. Stem surfaces of C. pulchra mirror the trichome make-up of the exterior floral tube surface.
164
Figure 7.6. Close-up scanning electron microscope image of a multicellular resin-secreting glandular trichome (TYPE 1) on the stem surface of Cuphea pulchra.
7.3.2 Verification of Cuphea pulchra Deterrence of Green Peach Aphid (Myzus persicae)
Settling by Choice and No-Choice Settling Assay
During the initial greenhouse observation period for this research it was noted that
C. pulchra displayed resistance to aphid settling, while other species housed in the same area were experiencing significant aphid infestations, particularly C. schumannii. In order to run a more controlled evaluation, a choice/no-choice assay was designed to evaluate the settling preference of green peach aphids. The results of this study represent the information and data of two experiments replicated in time.
165 Aphid counts were conducted two weeks after aphid introduction into the cages.
Comparison of species showed that when presented with the choice to settle on a C.
schumannii or a C. pulchra plant, significantly more (P<0.05) aphids settled on the C. schumannii plant (Figure 7.7). The mean number of aphids settled was approximately 7 and 21 per C. pulchra plant and 319 and 270 on C. schumannii for experiments one and
two respectively. Likewise, under no-choice settling conditions,
700 b 600 Exp. 2 500 Exp. 1
400
300
Total Aphids 200
100 a 0 C. pulchra C. schumannii
Figure 7.7. Comparison of mean number of total aphids settled per Cuphea pulchra or Cuphea schumannii plant under green peach aphid choice settling conditions. Each stacked column represents the means of experiments 1 and 2. Different letters above the stacked columns indicate a significant difference between species (P<0.05). Vertical bars indicate standard errors of the mean.
166 the number of aphids settled on C. schumannii plants was significantly higher (P<0.05) than the number of aphids settled on C. pulchra for both experiments conducted (Figure
7.8). C. schumannii averaged approximately 179 and 264 total aphids per plant to the 1 and 97 total aphids per C. pulchra plant for experiment one and two, respectively.
Species had a significant impact on aphid fecundity regardless or choice or no-choice treatment (Figure 7.9). The number of immatures on C. schumannii plants was significantly higher (P<0.05) than those found on C. pulchra, suggesting that fecundity corresponded with the adult preference to settle on C. schumannii.
550 Assay 2 Assay 1 b
. 450
350
250 Total Aphids Aphids Total
150 a
50 C. pulchra C. schumannii
Figure 7.8. Comparison of mean number of total aphids settled per Cuphea pulchra or Cuphea schumannii plant under green peach aphid no-choice settling conditions. Each stacked column represents the means of experiments 1 and 2. Different letters above the stacked columns indicate a significant difference between species (P<0.05). Vertical bars indicate standard errors or of the mean.
167 300
C. pulchra 250 C. schumannii b 200 b 150
Immature Aphid Immature 100
50 a a 0 Choice Plants No-Choice Plants
Figure 7.9. Mean number of immature aphids on C. pulchra and C. schumannii plants under choice or no- choice treatment conditions. The significantly higher number of immature aphids on C. schumannii reflects the higher aphid fecundity on this species over C. pulchra. Same letters above the column bars indicate that values are not significantly different (P<0.05). Vertical bars indicate standard errors of the mean.
Exposure to choice or no-choice settling conditions did not significantly affect
aphid settling or plant parameters for C. pulchra or C. schumannii (Table 7.2). No
significant difference existed between treatments for change in height or the number of
leaves expanded over the two week period, total number or lateral branches, number of
mature flowers, total aphids settled, leaf area of newly expanded leaves, or plant dry
weight for either species.
168
Leaf Area # Exp Newly Leaves Lateral Expanded Increase in Since Branches Total Leaves Total Dry 2 Height (cm) Initiation (>2cm) # Flowers Aphids (cm ) Wt. (g) C. pulchra 11.6 ± 2.5 31.5 ± 2.8 7.7 ± 1.5 5.7 ± 1.5 13.7 ± 6.4 185.0 ± 32.8 3.1 ± 0.7 (choice) 169 C. pulchra 13.0 ± 1.6 35.5 ± 4.6 7.5 ± 0.7 6.1 ± 1.4 49.4 ± 33.4 221.2 ± 37.7 3.4 ± 0.6 (no choice) C. schumannii (choice) 16.5 ± 3.2 7.3 ± 1.3 4.3 ± 1.3 0.8 ± 0.5 294.8 ± 84.3 230.5 ± 53.8 4.7 ± 1.2 C. schumannii
(no choice) 16.1 ± 1.9 8.4 ± 1.1 5.1 ± 1.0 1.5 ± 0.6 221.3 ± 55.1 237.0 ± 40.3 4.7 ± 0.8
Table 7.2. Treatment effect on Cuphea pulchra and Cuphea schumannii plant parameters. Maintenance under choice or no-choice aphid settling conditions did not significantly affect the change in height, number of expanded leaves, number of lateral branches, number of flowers, total aphids settled, leaf area, of total dry weight of either species after two weeks under respective conditions. Values are treatment means ± standard error. Increase in height was calculated by subtracting the initial height from the final height of the main stem after two weeks.
It is worthwhile to disclose at this point that a third replicate experiment was conducted between the two experiments discussed here. This experiment was unsuccessful due to the mortality of all aphids within one week of transfer to the experimental cages. However, all C. schumannii plants, regardless of choice or no- choice treatment were infested with thrips. Yet, there was no evidence of thrips on C. pulchra. This observation suggests that C. pulchra may maintain a natural resistance to thrips infestation as well as green peach aphid. Further study is required to confirm these observations and begin to identify possible mechanism of resistance, which may or may not be the same mechanism promoting aphid resistance.
Results of the whole-plant choice versus no-choice settling assays confirmed the initial greenhouse observations (discussed in chapter 2) that C. pulchra has the capability of deterring green peach aphid. Choice assays confirmed the high preference of aphid to settle on C. schumannii. The significantly low number of aphids settled on C. pulchra, especially under no-choice conditions suggested that the species maintains a resistance mechanism that deters or inhibits aphid settling on tissues to feed. This prompted further exploration into the mechanism of deterrence/resistance in the species.
7.3.3 The Influence of Glandular Trichome Exudate on Green Peach Aphid Settling
A major morphological difference between the two species under investigation in the research is the presence of glandular secreting trichomes on stem and floral tissues of
C. pulchra and the total lack thereof on plant surfaces of C. schumannii. An assay was designed to determine the role of trichome exudates on the settling of aphids on C.
170 pulchra plant tissues. If trichomes exudates do play a role in resistance, whether it is
mechanical or chemical in nature, the assumption is that removal of the exudates would
encourage increased settling of aphids.
After 24 and 48 hours, there was a significant difference in the number of aphids
settled on stem tissue and aphids settled on leaf tissue (Figure 7.10). Regardless of
whether or not the cutting had received an ethanol wash to remove stem trichome
exudates, the number of aphids on the leaves of cuttings was significantly higher than the
number of aphids found on the stems of cuttings. The importance of this finding lies in
the fact that C. pulchra does not possess glandular secreting trichomes on leaves but
does possess a high density of glandular secreting trichomes on stems. Thus, this result
indicates that the green peach aphid adults exhibited a preference to settle and bear their
live young on C. pulchra plant tissues lacking glandular trichomes.
The combined results of replicate assays suggests that removal of trichome
exudate droplets did not significantly influence the aphid settling over a 24 and 48 hour time period (Table 7.3). Alcohols (e.g. methanol or ethanol) were found to be effective in removing glandular trichome exudates from Solanum berthaultii leaf trichomes without causing major disruption to leaf tissues, however exudate droplets regenerated within 48 hours of removal (Neal et al., 1990). The lack of significance between the C.
pulchra cuttings washed with EtOH to removed exudates droplets and those cuttings that remained untreated may be due in part to the incomplete removal or the regeneration of trichome exudate droplets within the 48 hour assay period. This also suggests the possibility that trichome exudate may not be responsible for the apparent resistance of C. pulchra to green peach aphids.
171 12 Stem 10 Leaf b s 8 b
6
Number of Aphid of Number 4
2 a a
0 treated untreated
Figure 7.10. Mean number of aphids on stem and leaves of untreated C. pulchra cuttings and cuttings treated by an ethanol wash to remove trichome exudates droplets. Regardless of treatment, the number of aphids on the leaves of cuttings was significantly higher than the number of aphids on cutting stems. Same letters above the column bars indicate that values are not significantly different (P<0.05). Vertical bars indicate standard errors of the mean.
Aphids on Aphids on Total Aphids on Aphids on Total Stem Leaves Aphids Stem Leaves Aphid (24 hours) (24 hours) (24 hours) (48 hours) (48 hours) (48 hours) Treated (EtOH) 0.6 ± 0.2 4.3 ± 1.1 5.0 ± 1.1 1.3 ± 0.3 8.4 ± 2.1 9.8 ± 2.2
Control 0.9 ± 0.3 3.1 ± 0.8 4.1 ± 1.0 1.1 ± 0.4 7.4 ± 1.5 8.2 ± 1.7 No significant difference between treatements at P<0.05
Table 7.3. Mean number of aphids on C. pulchra cuttings washed with EtOH to remove trichome exudates droplets and untreated cuttings. Values are the treatment means ± standard error. No significant differences existed between treatments for total aphids settled, aphid settled on stems, and aphids settled on leaves 24 and 48 hours after aphid introduction.
172 7.3.4 Analysis of Cuphea pulchra Glandular Trichome Exudate
Results of green peach aphid settling assay provided a strong case for the significant role of glandular trichomes and the secreted exudates in the apparent resistance of C. pulchra. The question remained to be answered if the resistance induced by glandular trichome exudates is mechanical, meaning that the sticky resin limits the ability feed and settle, or if is a chemical resistance imposed by an exudate component toxic to aphids. Studies evaluating the resistance of Lycopersicon pennellii (Goffreda et al., 1989), Solanum berthaultii (Neal et al., 1990), tobacco sp. (Johnson et al., 2002), and petunia (Chortyk et al., 1997) to aphid and whitefly pests identified the primary causal agent for resistance in each of these species as a sugar ester in glandular trichome secretions. The similarities in aphid response in each of these studies to what was observed during settling assays prompted the exploration into the contribution of a sugar ester toward the resistance of C. pulchra.
Thin layer chromatography was used to determine if a sugar ester is present in the trichome exudates of C. pulchra stem tissue and thus evoking a chemical resistance to green peach aphid. This was done by running the sample against a sucrose octanoate standard. Sucrose octanoate is a sugar ester compound first found in the glandular trichomes of tobacco leaves (Puterka et al., 2003), which is currently being synthesized for use as an effective, biofriendly insecticide. The banding pattern produced by the C. pulchra sample was compared to that produced by the 1:10 dilution of the standard. TLC of the 1:10 dilution of the standard produced two discernable bands, a major band with a retention index (RI) of 0.06 and a minor band with a RI of 0.28 (Figure 7.11).
173 Column 1 Column 2 Column 3
Sucrose Standard C. pulchra C. pulchra Octanoate 1:10 Stem Wash Stem Wash Standard Dilution 1:10 Dilution
Figure 7.11. Digital photograph of the developed TLC plate comparing the banding pattern produced by the sucrose octanoate standard (column 1), a 1:10 dilution of the sucrose octanoate standard (column 2), and the stem wash sample of C. pulchra (column 3). Arrows indicate band development.
Presumably, the lower, slower moving band represents the various positional isomers of
the sucrose octanoate monoesters (W. Farone, Applied Power Concepts, personal
communication). In this analysis, the bands remaining near the start line are high in
sugar while polyester bands move up the plate furthest. Therefore, the sugar monoesters
will have the lowest retention index and the polyesters will have the highest retention
index. The undiluted Cuphea pulchra sample also produced two visible bands with the
major band co-chromatographing with the major band of the sucrose octanoate standard
174 with a RI of 0.06 (Figure 7.10). This, along with the same color response (blue-green) is taken as preliminary evidence that Cuphea pulchra stem trichome exudates do possess a sugar ester (sucrose octanoate or related compound) in which the primary acyl ester component is the same as that in a synthetic sucrose ester insecticide. Having only this primary information available, it is not possible to draw firm conclusions regarding the identity of the sugar ester of C. pulchra glandular trichome exudates. Additional analysis of trichome exudates is necessary to specifically identify the sugar as a sucrose or glucose and to identify the fatty acids esterified to the sugar.
7.4 Conclusions
Thorough assays evaluating aphid settling, it was possible to confirm preliminary greenhouse observations of C. pulchra resistance to the settling of green peach aphids.
Based on aphid preference to settle on C. pulchra leaf tissue lacking glandular secreting trichomes, over stem tissue, which does maintain a high density of glandular secreting trichomes, it is presumed that the apparent resistance of C. pulchra is due, at least in part, to the trichome exudate compounds.
Preliminary data from thin layer chromatography analysis indicates that the main component of the glandular trichome exudate of C. pulchra co-chromatographs with the main component(s) (presumably sugar monoesters) of the synthetic insecticide, sucrose octanoate. Further analysis is necessary to determine with certainty the identity of this compound. An approach to elucidating the identity of the presumed sugar ester of C. pulchra trichome exudates would be to fractionate C. pulchra stem tissue washes using high performance liquid chromatography (HPLC) to attain a crude sugar ester isolates,
175 which can be further purified using reverse-phase HPLC. At this point, purified samples can under-go two paths of analysis. First, TLC of the purified fractions would permit clearer and more precise banding, allowing for semiqualitative determination of sugar ester distribution in the sample as well as identifying isomers. Secondly, purified liquid chromatography fractions can be further analyzed by gas chromatography and mass spectrometry (GC-MS). This analysis would allow for the identification of the sugar
(sucrose or glucose) and the predominant fatty acid(s) esterified to the sugar. Positions of fatty acid esterification of sugar ester isomers may be determined through [1H] NMR spectra. This information would be necessary should there be cause to synthesize the active sugar ester of C. pulchra trichome exudate.
The question might arise as to why research with C. pulchra and its glandular trichome exudate should continue. This species presents a unique opportunity for the horticultural industry. Based on the performance of Cuphea pulchra in studies previously discussed in this research, this unique species holds much potential as a successful ornamental. The species inherent ability to deter insect pests is certainly a benefit for production, however, it may serve an even greater biochemical benefit to the industry. In a time when there is much concern about the greenhouse industry impact on the environment, it is important that research continues to find safe and effective alternatives to chemical insecticides. Synthetic sugar esters have already been developed and are being used as insecticidal compounds. However, further characterization of the physical and chemical properties of natural sugar ester compounds are needed to better
176 understand how sugar ester chemistry can be improved (Puterka et al., 2003). Cuphea pulchra provides a foothold for investigation into a genus that may have much to offer in terms unique trichome exudate chemistry.
177 CHAPTER 8
GENERAL CONCLUSIONS
The general objective of the research presented was to evaluate two wild species,
C. pulchra and C. schumannii for their potential as successful new introductions into the
ornamental horticulture industry. The evaluations and observations discussed in the
previous chapters provide the groundwork for the potential use as well as information
pertinent to commercial production of the crops.
Initial evaluation of C. schumannii suggests that this species holds little potential
as a successful ornamental plant. This species “leggy” and indeterminate growth habit
and high susceptibility to greenhouse pests and physiological disorder signals the
projected difficulty in successfully producing the plant under controlled environment
conditions. Observations of the species performance in the landscape reiterates the lack
of potential as the “leggy” growth habit persists and flowers fail to remain the focus as
the plant matures under outdoor conditions.
Evaluation of C. pulchra under production and outdoor conditions implies that the
species would perform successfully as a flowering annual crop in Midwestern climates.
Three lines of C. pulchra, “A”, “B”, and “C” and one mutant “white” were evaluated.
Based on performance under greenhouse and outdoor trial conditions, C. pulchra “A”
178 and “white” are the two that exhibit highest potential. C. pulchra “A” and its mutant
“white” maintain a rounded and compact growth habit with upright, “soft” branching,
which gives the plant a delicate appearance.
Based on the data collected in Columbus, OH, if C. pulchra was to be introduced
commercially, it would fit easily as a summer annual into a quarterly cropping schedule
of a Midwestern greenhouse grower. The start production date of a bedding annual is
based on the goal date for transplanting plants into the landscape or garden. For C.
pulchra, this date is late May, when there is no longer the threat of frost. Based on the
successful propagation schedule followed for the outdoor trials conducted for this
research, vegetative stem cuttings should be taken in mid to late March. Rooted cuttings
can be transplanted into 15.24 cm diameter pots approximately five weeks after cuttings
are taken from the stock plants.
Suggested production maintenance of the C. pulchra transplants is based on the
results of growth temperature, photoperiodic requirement, and growth regulation
evaluations discussed in chapters 4, 6, and 5 respectively. The species performed well
under daytime temperatures of approximately 21ºC to 25ºC and night temperature of
approximately 18ºC. Under these conditions, daily irrigation and fertilizing twice weekly
with a 200 mg N L-1 of 20-10-20 complete fertilizer is appropriate. C. pulchra responded
well to “pinching” or removal of the stem apex as a non-chemical method of growth
regulation with reduced internode elongation and increased lateral branch formation. The
unique flowers of C. pulchra are a highly marketable characteristic, therefore, production conditions should promote flowering. Because of the short production time of
179 approximately ten weeks for C. pulchra, transplanted rooted cuttings should be held under an inductive long day photoperiod to promote flowering. Based on observations of low susceptibility of the species to greenhouse insect pests and disease, use of chemical insecticides or fungicides should be minimal during production.
Simulated garden/landscape trials allowed for the collection of information important for labeling a flowering annual. C. pulchra performed very well when grown under conditions that included full sun and slightly acidic soils. C. pulchra “A” and
“white” reached a maximum height of 35 to 45 cm and therefore should be planted 40 to
60 cm apart. Both have sustained heavy production of flowers from late May through
October, which allows for the attraction of the broadest range of bee and hummingbird pollinators.
Cuphea pulchra not only has much to offer in terms of being an attractive addition to the floriculture industry, but its possession of a natural insect deterrence mechanism allows for the production of an ornamental crop with minimal chemical use.
The trichome exudate chemistry, sugar esters in particular, of C. pulchra provide a plausible mechanism of deterrence. The benefit of the sugar esters extends beyond the species itself, however. Because synthetic sugar esters have already been developed and are being used as insecticidal compounds, further characterization of natural sugar ester compounds are needed to better understand how sugar ester chemistry can be improved.
This research with C. pulchra certainly indicated the potential success of the species as an ornamental crop. However, it is important to reiterate that the evaluative studies for this species were conducted in Columbus, OH and illustrates the potential
180 success of the crop under similar climatic conditions. Extensive evaluation is necessary to determine the success of the crop under climatic conditions dissimilar to that of central
Ohio.
181 REFERENCES
Amarasinghe, V., S.A. Graham, and A. Graham. 1991. Trichome morphology in the genus Cuphea (Lythraceae). Botanical Gazette. 152(1): 77-90.
Aphalo, P.J., C.L. Bellare, and A.L. Scopel. 1999. Plant-plant signaling, the shade avoidance response and competition. Journal of Experimental Botany. 50 (340): 1629- 1634.
Armitage, A.M. 1986. Evaluation of new floricultural crops: a systems approach. Hortscience. 21(1): 9-11.
Armitage, A.M. 1988. New herbaceous ornamental crops research, P. 452-456. In: J. Janick and J.E. Simon (eds.), Advances in new crops: proceeding of the first national symposium new crops, research, development, and economics. Timber Press, Portland, OR.
Baskin, J.M. and C.C. Baskin. 1998. Ecology, Biogeography and Evolution of Dormancy and Germination. Academic Press, San Diego, CA.
Bernier, G., J. Kinet and R. Sachs. 1985. The Physiology of Flowering. CRC Press, Boca Raton, FL.
Bewley, J.D. and M. Black. 1994. Seeds: Physiology of Development and Germination. Plenum Press, New York, NY.
Chortyk, O.T., J.G. Pomonis, and A.W. Johnson. 1996. Syntheses and characterizations of insecticidal sucrose esters. Journal of Agricultural and Food Chemistry. 44: 1551- 1557.
Chortyk, O.T., S.J. Kays, and Q. Teng. 1997. Characterization of insecticidal sugar esters of Petunia. Journal of Agricultural and Food Chemistry. 45: 270-275.
Christie, P.J., M.R. Alfenito, and V. Walbot. 1994. Impact of low-temperature stress on general phenylpropanoid and anthocyanin pathways: Enhancement of transcript abundance and anthocyanin pigmentation in maize seedlings. Planta. 194: 541-549.
182 Clifford, S.C., E.S. Runkle, F.A. Langton, A. Mead. S. Foster, S. Pearson, and R. Heins. 2004. Height control of poinsettia using photoselective filters. Hortscience. 39(2): 383- 387.
Clough, E.A., A.C. Cameron, R.D. Heins, and W.H. Carlson. 2001. Growth and development of Oenothera fruticosa is influenced by vernalization duration, photoperiod, forcing temperature, and plant growth regulators. Journal of American Society for Horticultural Science. 126(3): 269-274.
Deitzer, G.F., R. Hayes, and M. Jabben. 1979. Kinetics and time dependence of the effect of far-red light on the photoperiodic induction of flowering in winter barley. Plant Physiology. 64: 1015-1021.
Dole, J.M. and H.F. Wilkins. 2005. Floriculture Principles and Species 2nd edition. Prentice Hall, Upper Saddle River, NJ.
Erwin, J.E. and R.D. Heins. 1991. Methods and schedules for forcing Easter lilies. Minn. Comm. Flow. Grow. Bull. 40(4): 1-18.
Farone, W. Applied Power Concepts. Anaheim, CA. Personal communication.
Frankland, B. 1986. Perception of light quantity, p. 219-236. In: R.E. Kendrick and G.H. Kronenberg (eds.) Photomorphogenesis in Plants. Martinus Nijhoff, Dordrecht.
Furuya, M. 1993. Phytochromes: their molecular species, gene families, and functions. Annual Review Plant Physiology and Plant Molecular Biology. 44: 617-645.
Gatehouse, J.A. 2002. Plant resistance towards insect herbivores: a dynamic interaction. New Physiologist. 156: 145-169.
Goffreda, J.C. M.A. Mutschler, D.A. Ave, W.M. Tingey, and J.C. Steffens. 1989. Aphid Deterrence by glucose esters in glandular trichome exudate of the wild tomato, Lycopersicon pennellii. Journal of Chemical Ecology. 15(7): 2135-2147.
Goffreda, J.C., E.J. Szymkowiak, I.M. Sussex, and M.A. Metschler. 1990. Chimeric tomato plants show that aphid resistance and triacylglucose production are epidermal autonomous characters. The Plant Cell. 2: 643-649.
Graham, S.A. 1988. Revision of Cuphea Section Heterodon (Lythraceae). Systematic Botany Monographs. 20.
Graham, S.A. 1990. New species of Cuphea section Melvilla (Lythraceae) and an annotated key to the section. Brittonia. 42(1): 12-32.
183 Graham, S.A. 1998. Revision of Cuphea section Diploptychia (Lythraceae). Systematic Botany Monographs. 53.
Halvey, A.H. 1999. New flower crops. In: J. Janick (ed.), Perspectives on new crops and new uses. ASHS Press, Alexandria, VA.
Harbone, J.B. and R.J. Grayer. 1988. The anthocyanins, 526-537. In: J.B. Harbone (ed.) The Flavinoids: Advances in Research Since 1980. Chapman and Hall Ltd., London.
Hatch, L.C. 1998. Why we need new ornamental plants. New Ornamentals Society web site. Raleigh, NC.
Heins, R. and J. Erwin. 1990. Understanding and applying DIF. Greenhouse Grower. 8(2): 73-78.
Henne, G. and S. Fakir. 1999. NBI-Ball agreement: a new phase in bioprospecting. Biotechnology and Development Monitor. 39: 18-21.
Hirsinger, F. and P.F. Knowles. 1984. Morphological and agronomic description of selected Cuphea germplasm. Economic Botany. 38: 439-451.
Hunt, E. Ball FloraPlant Greenhouse Operations Manager. Chicago, IL. Personal communication.
Jaworski, C.A., M.H. Bass, S.C. Phatak, and A.E. Thompson. 1988. Differences in leaf intumescence between Cuphea species. Hortscience. 23(5): 908-909.
Jaworski, C.A. and S.C. Phatak. 1991a. ‘Lavender Lady’, flowering ornamental Cuphea glutinosa. Hortscience. 26(2): 221-222.
Jaworski, C.A. and S.C. Phatak. 1991b. ‘Georgia Scarlet’, flowering ornamental Cuphea llavea. Hortscience. 26(10): 1343-1344.
Jaworski, C.A. and S.C. Phatak. 1992. Flowering ornamental Cuphea glutinosa ‘Purple Passion’ and ‘Lavender Lei’. Hortscience. 27(8): 940.
Johnson, A.W., V.A. Sisson, M.E. Snook, B.A. Fortum, and D.M. Jackson. 2002. Aphid resistance and leaf surface chemistry of sugar ester producing tobaccos. Journal of Entomologic. Science. 37(2): 154-165.
Kays, S.J., R.F. Severson, S.F. Nottingham, R.B. Chalfant, and O.T. Chortyk. 1994. Possible biopesticide from Petunia for the control of sweet potato whitefly (Bemisia tabaci) on vegetable crops. Proceedings of the Florida State Horticultural Society. 107: 163-167.
184 Kelsey, R.G., G.W. Reynolds, and E. Rodriguez. 1984. The chemistry of biologically active constituents secreted and stored in plant glandular trichomes, p. 188-244. In: E. Rodriquez, P. Healy, and I. Mehta (eds.) Biology and Chemistry of Plant Trichomes. Plenum Press, New York, NY.
Knapp, S.J. 1989. New temperate industrial oilseed crops, p. In: J. Janick and J. Smith (eds.) First National New Crops Symposium. Timber Press, Portland, OR.
Knapp, S.J. and L.A. Tagliani. 1989. Genetic variation for seed dormancy in Cuphea laminuligera and Cuphea lanceolata. Euphytica. 47: 65-70.
Knapp, S.J. 1990. Recurrent mass selection for reduced seed dormancy in Cuphea lanceolata and Cuphea laminuligera. Plant Breeding. 104: 46-52. Lang, A. 1965. Physiology of flower initiation, p.1380-1536. In: W. Ruhland (ed.), Encyclopedia of Plant Physiology. Springer-Verlag, Berlin.
Lang, A., M. Chailakhyan, and I. Frolova. 1977. Promotion and inhibition of flower formation in a day neutral plant in grafts with a short-day plant and a long-day plant. Proceedings of the National Academy of Science USA. 74: 2412-2516.
Lang, S.P., B.E. Struckmeyer, and T.W. Tibbitts. 1983. Morphology and anatomy of intumescence development on tomato plants. Journal of the American Society of Horticultural Science. 108(2): 266-271.
Lang, S.P. and T.W. Tibbitts. 1983. Factors controlling intumescence development on tomato plants. Journal of the American Society of Horticultural Science. 108(1): 93-98.
Leopold, J.H., J.D. Metzger, and S.A. Graham. 2003. Development and evaluation of Cuphea pulchra and Cuphea schumannii as new landscape ornamentals. ISHS ACTA Horticulturae. 624: 85-91.
Levin, D.A. 1973. The role of trichomes in plant defense. Quarterly Review of Biology. 48: 3-15.
McMahon, M.J. and J.W. Kelly. 1990. Influence of spectral filters on height, leaf chlorophyll, and flowering of Rosa x hybrida ‘Meirutral’. Journal of Environmental Horticulture. 8(4): 209-211.
McMahon, M.J., J.W. Kelly, D.R. Decoteau, R.E. Young, and R.K. Pollock. 1991. Growth of Denranthema x grandiflorum (Ramat.) Kitamura under various spectral filters. Journal of American Society for Horticultural Science. 116: 950-954.
McMahon, M.J. 1999. Development of chrysanthemum meristems grown under far-red absorbing filters and long or short photoperiods. Journal of American Society for Horticultural Science. 214(5): 483-487.
185
Mello, M.O. and M.C. Silva-Filho. 2002. Plant-insect interactions: an evolutionary arms race between two distinct defense mechanisms. Brazilian Journal of Plant Physiology. 14(2): 71-81.
Metwally, A.W., B.E. Struckmeyer, and G.E. Beck. 1970a. Effect of three soil moisture regimes on the growth and anatomy of Pelargonium hortorum. Journal of American Society for Horticultural Science. 95(6): 803-808.
Metwally, A.W., G.E. Beck, and B.E. Struckmeyer, 1970b. The role of water and cultural practices on oedema of Pelargonium hortorum Ait. Journal of American Society for Horticultural Science. 95(6): 808-813.
Moe, R. 1993. Control of plant morphogenesis and flowering by temperature alterations. FLN. 15: 30-34.
Moe, R. and R.D. Heins. 1990. Control of plant morphogenesis and flowering by light quality and temperature. Acta Horticulturae. 272: 81-89.
Moe, R., N. Glomsrud, I. Bratberg, and S. Valso. 1992. Control of plant height in poinsettia by temperature drop and graphical tracking. Acta Horticulturae. 327: 41-48.
Morrow, R.C. and T.W. Tibbitts. 1987. Induction of intumescence injury on leaf disks. Journal of the American Society for Horticultural Science. 112(2): 304-306.
Myster, J. and R. Moe. 1995. Effect of diurnal temperature alterations on plant morphology in some greenhouse crops- a mini review. Scientia Horticulturae. 62: 205- 215.
National Botanical Institute (NBI) of South Africa. 2001. The NBI-Ball agreement and bioprospecting in perspective. NBI web site.
Neal, J.J., W.M. Tingey, and J.C. Steffens. 1990. Sucrose esters of carboxylic acids in glandular trichomes of Solanum berthaultii deter settling and probing by green peach aphid. Journal of Chemical Ecology. 16(2): 487-497.
Nelson, P.V. 1988. Greenhouse Operation and Management (5th edition). Prentice Hall, Upper Saddle River, NJ.
Nissim-Levi, A., S. Kagan, R. Ovadia, and M. Oren-Shamir. 2003. Effects of temperature, UV-light, and magnesium on anthocyanin pigmentation in cocoplum leaves. Journal of Horticultural Science & Biotechnology. 78(1): 61-64.
O’Neil, S. 1992. The photoperiodic control of flowering: progress toward understanding the mechanism of induction. Photochemistry and Photobiology. 56(5): 789-801.
186
Oren-Shamir, M. and A. Levi-Nissim. 1997. Temperature effect on the leaf pigmentation of Contius coggygria ‘Royal Purple’. Journal of Horticultural Science. 72:425-432.
Oyaert, E., E. Volckaert, and P.C. Debergh. 1999. Growth of chrysanthemum under coloured plastic films with different light qualities and quantities. Scientia Horticulturae. 79: 195-205.
Pearson, S., A. Parker, P. Hadley, and H.M. Kitchner. 1995. The effect of photoperiod and temperature on reproductive development of cape daisy (Osteospermum jucundum cv. ‘Pink Whirls’. Scientia Horticulturae. 62: 225-235.
Perez-Castorena, A.L. and E. Maldonado. 2003. Triterpenes and flavonoid glycosides from Cuphea wrightii. Biochemical Systematics and Ecology. 31: 331-334.
Puterka, G.J., W. Farone, T. Palmer, and A. Barrington. 2003. Structure-function relationship affecting the insecticidal and miticidal activity of sugar esters. Journal of Economic Entomology. 96(3): 636-644.
Rademacher, W. 2000. Growth Retardants: Effect of gibberellin biosynthesis and other metabolic pathways, p.501-531. In: R.L. Jones (ed.) Annual Review of Plant Physiology and Plant Molecular Biology. Annual Reviews, Palo Alto, CA.
Rajapakse, N.C., R.E. Young, M.J. McMahon, and R. Oi. 1999. Plant height control by photoselective filters: current status and future prospects. HortTechnology. 9(4): 618- 624.
Raven, P.H., R.F. Evert, and S.E. Eichhorn. 1999. Biology of Plants (6th Edition). W.H. Freemen Co., New York, NY.
Roh, M.S. and R.H. Lawson. 1988. New floriculture crops, p.448-453. In: J. Janick and J.E. Simon (eds.), Advances in new crops: proceeding of the first national symposium new crops, research, development, and economics. Timber Press, Portland, OR.
Roh, M.S. and R.H. Lawson. 1996. New floral crops in the United States. p.526-535. In: J. Janick (ed.), Progress in new crops. ASHS Press, Arlington, VA.
Rojas, M. 1999. The biotrade initiative: programme for biodiversity-based development. Biotechnology and Development Monitor. 38: 11-14.
Santos, D.Y.A., M.L.F. Salatino, and A. Salatino. 1995. Favonoids of species of Cuphea (Lythraceae) from Brazil. Biochemical Systematics and Ecology. 23(1): 99- 103.
187 Seabrook, J.E. and L.K. Douglass. 1998. Prevention of stem growth and inhibition and alleviation of intumescence formation in potato plantlets in vitro by yellow filters.
Secretariat of the Convention of Biological Diversity. 2001. Biodiversity-the web of life. Montreal, Quebec Canada.
Seiler, A. and G. Dutfield. 2002. Regulating access and benefit sharing. Biotechnology and development monitor. 49: 3-7.
Severson, R.F., D.M. Jackson, A.W. Johnson, V.A. Sisson, and M.G. Stephenson. 1991. Oviposition behavior of tobacco budworm and tobacco hornworm: effects of cuticular components from Nicotiana species. Am. Chem. Soc. Ser. 449:264-277.
Suzuki, A. and J.D. Metzger. 2001. Vernalization in a greenhouse promotes and synchronizes flowering of Osteospermum ecklonis Norl. Hortscience. 36(4): 658-660.
Taiz, L. and E. Zeiger. 1998. Plant Physiology (2nd edition). Sinauer Associates, Inc., Sunderland, MA.
Thomas, B. and D. Vince-Prue. 1997. Photoperiodism in Plants. Academic Press. San Diego, CA.
Thompson, A.E. 1985. New native crops for the arid southwest. Economic Botany. 39(4): 436-453.
Thompson, A.E., W.W. Roath, and M.P. Widrlechner. 1995. ‘Starfire’ Cuphea hybrid. Hortscience. 30(1): 166-167.
United States Department of Agriculture and National Agricultural Statistics Services. 2004. Floriculture crops 2003 summary (online).
Vierstra, R.D. 1993. Illuminating Phytochrome function. Plant Physiology. 103:679- 684.
Wagner, G.J. 1991. Secreting glandular trichome: more than just hairs. Plant Physiology. 96: 675-679.
Widrlechner, M.P. and D.A. Kovach. 2000. Dormancy-breaking protocols for Cuphea seed. Seed Science and Technology. 28: 11-27.
Zar, J.H. 1974. Biostatistical Analysis. Prentice Hall, Englewood Cliffs, NJ.
Zeevaart, J. 1969. Perilla, p.116-155. In: L.T. Evans (ed.), Induction of flowering: some case histories. Cornell University Press, Ithaca, NY.
188