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A study on the propagation methods for two native wildflowers

Permanent Link: http://ufdc.ufl.edu/UFE0041331/00001

Material Information

Title: A study on the propagation methods for two native wildflowers Polygonella polygama and Polygonella robusta.
Physical Description: 1 online resource (95 p.)
Language: english
Creator: Heather, Alison
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: dormancy, gibberellin, physiological, stratification
Environmental Horticulture -- Dissertations, Academic -- UF
Genre: Horticultural Science thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The perennial nature, prolific white to pink flower racemes, and attractive foliage and form of October flower (Polygonella polygama (Vent.) Engelm. & A. Gray Polygonaceae) and sandhill wireweed (Polygonella robusta (Small) G.L. Nesom & V.M. Bates Polygonaceae) suggest that these wildflowers could have significant ornamental and landscape potential if an effective propagation method can be developed. Prior to germination experiments, both species were tested for viability using Triphenyltetrazolium chloride (TZ). Tests indicate the seed viability was variable (P. polygama= 77%, P. robusta= 54%). Initial germination tests showed that both species germinated best in cooler temperatures (22oC day and 11oC night temperatures). Both species exhibited physiological dormancy. Germination was greatest when seeds were treated with 100 or 1000ppm GA3 (P. polygama = 35%, P. robusta = 58%) and kept in an incubator at 22/11 oC. Germination of both species at simulated seasonal temperatures, carried out in a move-along experiment, indicated that seeds require a period of warm stratification before germination commences at cooler temperatures. Additionally, P. polygama seeds stratified in 5oC for 2 weeks prior to planting out in the greenhouse showed an improvement in germination. However, poor collection timing and improper post-harvest storage may decrease germination. Propagation by stem cuttings may decrease production time, improve uniformity, and widen collection times. Experiments were conducted to determine the effects of Indole-3-butyric acid (IBA) and 1-Naphthaleneacetic acid (NAA) on rooting softwood cuttings of P. polygama and P. robusta collected from natural populations in central and south Florida. Softwood cuttings of each species were quick dipped with nine different concentrations of K-IBA:K-NAA (0:0, 0:250, 0:500, 500:0, 500:250, 500:500, 1000:0, 1000:250, 1000:500 ppm). Root initiation and quality were assessed after 6 weeks (P. polygama) or 8 weeks (P. robusta) under intermittent mist. When P. polygama (south) cuttings were treated with 1000:250 IBA:NAA ppm, rooting reached 63%. The highest rooting of 80% was achieved for P. robusta (south) when treated with 500:250 IBA:NAA ppm. Significant site times IBA times NAA interactions occurred for root index and percent rooting of P. robusta. However, most measured responses were not significantly different among auxin treatments.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Alison Heather.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Perez, Hector.
Local: Co-adviser: Wilson, Sandra B.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-06-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0041331:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041331/00001

Material Information

Title: A study on the propagation methods for two native wildflowers Polygonella polygama and Polygonella robusta.
Physical Description: 1 online resource (95 p.)
Language: english
Creator: Heather, Alison
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: dormancy, gibberellin, physiological, stratification
Environmental Horticulture -- Dissertations, Academic -- UF
Genre: Horticultural Science thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The perennial nature, prolific white to pink flower racemes, and attractive foliage and form of October flower (Polygonella polygama (Vent.) Engelm. & A. Gray Polygonaceae) and sandhill wireweed (Polygonella robusta (Small) G.L. Nesom & V.M. Bates Polygonaceae) suggest that these wildflowers could have significant ornamental and landscape potential if an effective propagation method can be developed. Prior to germination experiments, both species were tested for viability using Triphenyltetrazolium chloride (TZ). Tests indicate the seed viability was variable (P. polygama= 77%, P. robusta= 54%). Initial germination tests showed that both species germinated best in cooler temperatures (22oC day and 11oC night temperatures). Both species exhibited physiological dormancy. Germination was greatest when seeds were treated with 100 or 1000ppm GA3 (P. polygama = 35%, P. robusta = 58%) and kept in an incubator at 22/11 oC. Germination of both species at simulated seasonal temperatures, carried out in a move-along experiment, indicated that seeds require a period of warm stratification before germination commences at cooler temperatures. Additionally, P. polygama seeds stratified in 5oC for 2 weeks prior to planting out in the greenhouse showed an improvement in germination. However, poor collection timing and improper post-harvest storage may decrease germination. Propagation by stem cuttings may decrease production time, improve uniformity, and widen collection times. Experiments were conducted to determine the effects of Indole-3-butyric acid (IBA) and 1-Naphthaleneacetic acid (NAA) on rooting softwood cuttings of P. polygama and P. robusta collected from natural populations in central and south Florida. Softwood cuttings of each species were quick dipped with nine different concentrations of K-IBA:K-NAA (0:0, 0:250, 0:500, 500:0, 500:250, 500:500, 1000:0, 1000:250, 1000:500 ppm). Root initiation and quality were assessed after 6 weeks (P. polygama) or 8 weeks (P. robusta) under intermittent mist. When P. polygama (south) cuttings were treated with 1000:250 IBA:NAA ppm, rooting reached 63%. The highest rooting of 80% was achieved for P. robusta (south) when treated with 500:250 IBA:NAA ppm. Significant site times IBA times NAA interactions occurred for root index and percent rooting of P. robusta. However, most measured responses were not significantly different among auxin treatments.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Alison Heather.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Perez, Hector.
Local: Co-adviser: Wilson, Sandra B.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-06-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0041331:00001


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A STUDY ON PROPAGATION METHOD S FOR TWO NATIVE WILDFLOWERS: POLYGONELLA POLYGAMA AND POLYGONELLA ROBUSTA BY ALISON HEATHER A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORID A IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2009 1

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2009 Alison Heather 2

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To everyone who has helped me with this pr oject, even if it was just a word of encouragement 3

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ACKNOWLEDGMENTS I thank my parents for their patienc e and encouragement in helping me become who I am today. I greatly appreciate the time that was taken by my advisors Dr. Hector Perez and Dr. Sandy Wilson, who developed me into the hopefully knowledgeable graduate student I have become. I thank the Florida Stat e Wildflower Council who helped partially fund this study. I also would like to thank the following people for their contributions: my committee members Dr. Mack Thetford and Dr. Debbie Miller, Fe Almira for her kind words and unceasing l aboratory and greenhouse support; the UF ICBR Lab for assistance with microscopy work; Steve Woodmansee for his dedicated seed collection; Carolyn Bartuska for her aide and patience with statistics; and Dominique Ardura, Julia Stover Keona Muller, and Pat Frey fo r their help with lab work. I greatly appreciate the thankless editi ng and moral support of my fianc Patrick ODonoughue. Also, a special thanks to the Department of Environmental Horticulture and the University of Florida. 4

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TABLE OF CONTENTS page ACKNOWLEDG MENTS..................................................................................................4 LIST OF TABLES............................................................................................................7 LIST OF FI GURES ..........................................................................................................8 ABSTRACT.....................................................................................................................9 CHAPTER 1 INTRODUCTION AND LI TERATURE REVIEW.....................................................11 History of Florida and Native Plants........................................................................11 Economics of Florida Native Pl ants........................................................................12 Ecological Benefits of Fl orida Native Plants............................................................13 Native Wild flowers..................................................................................................14 Native Wildflower s of Flor ida..................................................................................15 Landscape Potential of Two Flor ida Native W ildflower s.........................................17 Propagation by Seed..............................................................................................19 Propagation by Cuttings ..........................................................................................22 Experiment Pr eface................................................................................................26 2 GERMINATION AND DORMANCY STUDIES OF POLYGONELLA POLYGAMA AND POLYGONELLA ROBUSTA ..........................................................................29 Introducti on.............................................................................................................29 Materials and Methods............................................................................................32 Seed Collection and Stor age............................................................................32 Pre-Germination Vi ability Assay.......................................................................33 Initial Germinat ion Test s...................................................................................33 Water Uptake by Intact and Scarifi ed Seeds ....................................................34 Seed Anatomy and Morphology.......................................................................35 Requirements for Warm or Cold Strati ficati on..................................................36 Effect of Cold Stratification on Se eds Propagated in t he Greenhouse.............37 Effects of Gibberellic Acid on Se eds Propagated in the Laboratory and Greenhouse ..................................................................................................37 Initial Seedling Emergence and the Effe ct of Different Propagation Media......38 Data Anal ysis...................................................................................................39 Result s....................................................................................................................39 Pre-Germination Viability Assay and Initial Germina tion Tests.........................39 Water Uptake by Intact and Scarifi ed Seeds ....................................................40 Seed Anatomy and Morphology.......................................................................41 Requirements for Warm or Cold Strati ficati on..................................................41 Effect of Cold Stratification on Se eds Propagated in t he Greenhouse.............42 5

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Effects of Gibberellic Acid on Se eds Propagated in the Laboratory and Greenhouse ..................................................................................................43 Initial Seedling Emergence and the Effe ct of Different Propagation Media......44 Discussio n..............................................................................................................45 3 EFFECT OF AUXIN APPLICAT ION ON ROOTIN G OF SOFTWOOD CUTTINGS OF POLYGONELLA POLYGAMA AND POLYGONELLA ROBUSTA ..............................................................................................................63 Introducti on.............................................................................................................63 Materials and Methods............................................................................................65 Cutting source and aux in treatments................................................................65 Data Coll ection.................................................................................................66 Experimental Design and Data An alysis ..........................................................67 Result s....................................................................................................................67 Polygonella polygama ......................................................................................67 Polygonella robusta ..........................................................................................69 Discussio n..............................................................................................................71 4 CONCLUS IONS.....................................................................................................85 LIST OF RE FERENCES...............................................................................................88 BIOGRAPHICAL SKETCH ............................................................................................95 6

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LIST OF TABLES Table page 2-1 Change in temperatures detailed in the move-along experiment with number of weeks seeds spent in inc ubation at each te mperatur e...................................52 2-2 Pre-germination viability of all seed sources tested using 1% TZ solution.........53 2-3 Germination and TZ viability testing of Polygonella polygama (C) and Polygonella robusta seeds.................................................................................53 2-4 Post-germination viabilit y from the dark treatment at all temperatures tested using a 1% TZ solution .......................................................................................54 2-5 Analysis of variance table of emergence percent and rate for class comparison between sterilizat ion and media treatments of P. polygama (N).....54 3-1 Collection date, site location, and site information for cuttings of Polygonella polygama (October flower) and Polygonella robusta (sandhill wireweed)..........74 3-2 Collection site charac teristics for cuttings of Polygonella polygama (October flower) and Polygonella robusta (sandhill wireweed)..........................................74 3-3 Cuttings of October flower ( Polygonella polygama ) and sandhill wireweed ( Polygonella robusta ) were quick dipped in one of the following indole-3butyric acid (IBA) 1-naphthalen eacetic acid (N AA) used ....................................75 3-4 Effects of K-IBA and K-NAA treat ments on rooting softwood cuttings of October flower ( Polygonella polygama ) collected from central or south Florida popu lations .............................................................................................76 3-5 Effects of K-IBA and K-NAA treat ments on rooting softwood cuttings of Sandhill wireweed ( Polygonella robusta ) collected from central or south Florida popu lations .............................................................................................77 3-6 Trend analysis of P. polygama root index. Main effe cts equaled the source locations .............................................................................................................78 3-7 Trend analysis of P. robusta root index..............................................................78 3-8 Trend analysis of P. robusta percent r ooting. .....................................................78 3-9 Trend analysis of P. robusta root num ber...........................................................79 3-10 Trend analysis of P. robusta root l ength.............................................................79 7

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LIST OF FIGURES Figure page 1-1 Differences in the morphological characteristics and distribution of Polygonella polygama and Polygonella robusta .................................................27 2-1 Emergence percent as an effect of media types and surface sterilization on seeds of P. polygama (N)...................................................................................55 2-2 Increase in fresh mass of scari fied or non-scarified seeds of P. polygama or P. robusta ...........................................................................................................56 2-3 Thick sections of Polygonella seeds...................................................................57 2-4 Cumulative germination of seeds collected in central Florida of P. polygama and P. robusta incubated at simulated seasonal temper atures..........................58 2-5 Final emergence percent and mean germination time of P. polygama (N) seeds after moist stratification in the dark at 5 oC for 0, 2, 4, or 8 wk.................59 2-6 Final germination percent and mean germination time of P. polygama (C) seeds after soaking for 24 h in varying concentrations of GA3 and subsequent incubation at the constant temperature of 22/11 oC (72/52 oF) for 28 days. ..............................................................................................................60 2-7 Final germination percent and mean germination time of P. robusta seeds after soaking for 24 h in varying concentrations of GA3 and subsequent incubation at the constant temperature of 22/11 oC (72/52 oF) for 28 days........61 2-8 Final emergence percent and mean germination time of P. polygama (N) seeds after soaking for 24 h in varying concentrations of GA3 and subsequent growth in the misthouse fo r 42 da ys................................................62 3-1 Representation of 5 root index categories. ........................................................80 3-2 Representative central Florida P. polygama cuttings 8 wk after being quick dipped in various rooting hormone concent rations.............................................81 3-3 Representative south Florida P. polygama cuttings 8 wk after being quick dipped in various rooting hormone concent rations.............................................82 3-4 Representative central Florida P. robusta cuttings 6 wk after being quick dipped in various rooting hormone concent rations.............................................83 3-5 Representative south Florida P. robusta cuttings 6 wk after being quick dipped in various rooting hormone concent rations.............................................84 8

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Abstract of Thesis Pres ented to the Graduate School of the University of Florida in Partial Fulf illment of the Requirements for t he Degree of Master of Science PROPAGATION METHODS OF TWO NATIVE WILDFOWERS: POLYGONELLA POLYGAMA AND POLYGONELLA ROBUSTA By Alison Heather December 2009 Chair: Hector E. Perez Cochair: Sandra B. Wilson Major: Horticultural Science The perennial nature, prolific white to pink flower racemes, and attractive foliage and form of October flower (Polygonella polygama (Vent.) Engelm. & A. Gray [Polygonaceae]) and sandhill wireweed ( Polygonella robusta (Small) G.L. Nesom & V.M. Bates [Polygonaceae]) suggest that t hese wildflowers could have significant ornamental and landscape potential if an effective propagation method can be developed. Prior to germination experiments, both species were tested for viability using Triphenyltetrazolium chloride (TZ). Tests indicate the seed viability was variable ( P. polygama= 77%, P. robusta= 54%). Initial germination test s showed that both species germinated best in cooler temperatures (22oC day and 11oC night temperatures). Both species exhibited physiological dormancy. Germination was greatest when seeds were treated with 100 or 1000ppm GA3 (P. polygama = 35%, P. robusta = 58%) and kept in an incubator at 22/11 oC. Germination of both species at simulated seasonal temperatures, carried out in a move-along experim ent, indicated that seeds require a period of warm stratification before germinat ion commences at cooler temperatures. Additionally, P. polygama seeds stratified in 5oC for 2 weeks prior to planting out in the 9

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10 greenhouse showed an improvement in germinat ion. However, poor collection timing and improper post-harvest storag e may decrease germination. Propagation by stem cuttings may decreas e production time, improve uniformity, and widen collection times. Experiments were conducted to determine the effects of Indole-3-butyric acid (IBA) and 1-Naphthaleneacetic acid (NAA) on rooting softwood cuttings of P. polygama and P. robusta collected from natural populations in central and south Florida. Softwood cuttings of each spec ies were quick dipped with nine different concentrations of K-IBA:K-NAA (0:0, 0: 250, 0:500, 500:0, 500: 250, 500:500, 1000:0, 1000:250, 1000:500 ppm). Root initiation and quality were assessed after 6 weeks (P. polygama ) or 8 weeks ( P. robusta ) under intermittent mist. When P. polygama (south) cuttings were treated with 1000: 250 IBA:NAA ppm, rooting r eached 63%. The highest rooting of 80% was achieved for P. robusta (south) when treated with 500:250 IBA:NAA ppm. Significant site IBA NAA interact ions occurred for root index and percent rooting of P. robusta However, most measured res ponses were not significantly different among auxin treatments.

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CHAPTER 1 INTRODUCTION AND LI TERATURE REVIEW History of Florida and Native Plants Florida has been inhabited by Europeans and their descendents since 1565, with their impact being fully visible with the cession of Florida to England in 1763 (Myers and Ewel, 1990). Though Native Americans had lived in Florida for centuries prior to European settlement, their impact on the state was less severe compared to the changes seen today. Though Native Americans used fire to clear understories for agriculture and improve passage, thus mimicking the effects of natural wildfire, they also first began the practice of fi re suppression (Delcourt, 1987). Landscapes where fire has been suppressed may have higher intensity wildfires due to fuel accumulation, preventing the beneficial postburn effects that most ecosystems are adapted to. The arrival of European settlers marked the beginni ng of the drastic transformation of the Florida landscape, including a lack of underst anding of fire. Since the settlers arrival there have been three major events which have further altered ecosystems of Florida: ubiquitous clearing of land for agriculture and silviculture to support the expanding population, conversion of 80% of Lake Wales ridge for t he cultivation of citrus (Christman and Judd, 1990), and attempt ed drainage of Lake Okeechobee and the Everglades (Myers and Ewel, 1990). Since civilization has entered Florida, urban development has taken over hal f of Floridas pine flatwoods (Taylor, 1998). The scrub ecosystem, which is home to many wildfl owers, has suffered great losses. There are many ways to define a native plant; the U.S. Fish and Wildlife Service (2001) considers a species native to a particu lar ecosystem if other than a result of introduction, (it has) historically occurred or currently occurs in that ecosystem. The 11

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Florida Native Plant Society (F NPS) specifies that a plant is native to Florida if it has occurred within the state s boundary before Eur opean contact (FNPS, 2003). In 1992 it was estimated that there were approximately 2,870 plants native to Florida (Marinelli, 1994). Wunderlin and Hansen (2003) list 4,200 s pecies of native or naturalized ferns and seed plants in their Atlas of Florida Vascular Plants, making Florida the third most floristically diverse state in the United St ates. Floridas distinctive native flora has multiple origins. Plants occurring in South Florida are often also found in the Bahamas, West Indies, and South America. Those from North Florida can often be found in Texas and throughout the Midwest and Appalachian Mountains. Florida also possesses many endemic native plants. An endemic plant is unique to a particular region and can be a measure of species richness in a given ar ea (Taylor, 1998). For example, the Central Florida Ridge is a unique area of relict Pleistocene epoch dunes composed primarily of scrub ecosystem. Forty species of endemic plant s occur in this region, thus providing one of the highest rates of endemism in No rth America (Christman and Judd, 1990). Economics of Florida Native Plants In 2005, sales in the Florida environmental horticulture sector reached over $15 billion. Of these sales, 10.5% were native species (Hodges and Haydu, 2006), an increase of 3.5% since 1997 (Hodges and Haydu, 1999). The popularity of native plants appears to be increasing as evidenced by growing sales, establishm ent of more native plant society chapters (FNPS, 2003) and wider plant ava ilability listed by the Association of Florida Native Nurseries (AFNN, 2009). In addition, there are several books that have been written in recent years that concentrate only on Florida native landscaping (Walton and Schiller, 2007; Os orio, 2001; Haehle and Brookwell, 1999; Huegel, 1995). Moreover, groups that spec ifically focus on wildflowers have been 12

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formed such as the Florida Wildflower F oundation and the Wildfl ower Seed and Plant Growers Association, Inc. The Florida Wildfl ower Foundations mission is to protect and replenish native wildflowers while increasing public knowledge of them as vital members of the states delicately balanced ecosystems (Florida W ildflower Foundation, 2009). Currently the Wildflower Seed and Plant Gr owers Association, Inc. works with the Florida Wildflower Foundation to popularize the production of wildflowers with over a dozen active seed producers across the stat e (Wildflower Seed and Plant Growers Association, Inc., 2003; J. Norc ini, personal communication). Ecological Benefits of Florida Native Plants Native plants attract a wide variety of bi rds and insects. Observational studies indicate that alien plants support fewer insect species than natives (Tallamy, 2004). Ornamentals commonly promoted by the horticulture industry are selected because they are pest free (Mack and Erneberg, 2002). A decline in the number of insect species will invariably cause the decline of insectivores, which could continue through the food web (Tallamy, 2004). As Huegel (1995) simply put native plants and diversity of wildlife are intertwined. Wildlife us e plants for both food and cover (Huegel, 1995). An example of this dependency is the gopher apple ( Licania michauxii [Chrysobalanaceae]), which received its co mmon name because of the nourishment it provides to the gopher tortoise. Furthermo re, creating a landsc ape enriched with native plants can promote genetic diversity and reduc e habitat fragmentation (Marinelli, 1994). Although Scheiber et al. (2008) found no difference in the amount of water required for postestablishment growth betwe en native and exotic shrubs, anecdotal evidence suggests that Florida native plant s that are properly selected require less water, fertilizer, and pesticides than non-natives This conclusion is supported by the 13

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knowledge that native pl ants adapt to the nutrient poor soils, unique rainfall patterns and intense sunlight (Walton and Schiller 2007; Haehle and Brookwell, 1999). Native Wildflowers The National Wildlife Resear ch Center describes a wildflower as a flowering plant that is native to a specific geographical ar ea or habitat, capable of growing without the assistance of humans (Paulson, 1989). Therefore, once an area that fits the plants desired condition is seeded with wildflowers, it should be maintained with little effort (Paulson, 1989; Phillips, 1985). Florida nativ e wildflowers often thrive in the hot and humid summers found in the state, unlike mo st industry termed perennials which often dont survive more than two years (Walton and Schiller, 2007; Harper-Lore and Wilson, 2000). The wildflower industry boom began in 198 2 with Lady Bird Johnsons opening of the National Wildflower Research Center in Austin, Texas (Milstein, 2005). Since then, interest in wildflowers has spread to highway departments, homeowners, and restoration specialists. Wildflowers are chosen for landscape use because they are easy to cultivate, often showy perennials (Miles, 1976). Wildflow ers have primarily been used for roadside beautification and erosion control (Milstein, 2005). Considering this niche, the most widely utilized wildflower s pecies have been relatively inexpensive, easy to grow, and adaptable to a wide range of habitats and conditions (Milstein, 2005). In cultivation, wildflowers often flourish due to lack of competit ion from neighboring plant species (Phillips, 1985). Hammond et al. (2007) reported that firewheel ( Gaillardia pulchella [Asteraceae]) grown individually in landscape plots were up to 2.5 times larger than those found in their native habitat. Fu rther, Frances (2008) found reduced above and below ground biomass in tickseed ( Coreopsis spp. [Asteraceae]) when planted in 14

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pots with other tickseed plants or bahiagrass ( Paspalum spp. [Poaceae]). In this study, intraspecific competition decreased biomas s more than interspecific competition (Frances, 2008). Weed competition is one prim ary reason wildflowers do not establish within a year or two of plant ing (Norcini and Aldrich, 2004) Native plants are also commonly less aggressive, which makes them more susceptible to weed intrusions (Pfaff et al., 2002). Native Wildflowers of Florida Wildflowers exist in most Florida eco systems. They dominate the understory of pine flatwoods and can be found on open sandhills and sand pine scrub (Taylor, 1998). Harsh environments such as flatwoods and dry prairies can support a diverse understory with wildflowers such as tarflower ( Befaria racemosa [Ericaceae]), yellow bachelors button ( Polygala rugelii [Polygalaceae]), fall-flowering ixia ( Nemastylis floridana [Iridaceae]), and scare-weed ( Baptisia simplicifolia [Fabaceae]) (Myers and Ewel, 1990). Scrubs and sandhills, that usua lly consist of soils of the order entisol derived from quartz sand, generally have a more sparse ground cover that includes species such as gopher apple ( Licania michauxii [Chyrsobalanceae]), milk peas ( Galactia spp. [Fabaceae]), and garberia ( Garberia heterophylla [Asteraceae]) (Myers and Ewel, 1990). Fire is a vital component in maintaining the stability of many Florida ecosystems. In the absence of fire due to intentional fire suppression or habitat fragmentation, the ecosystem is disrupt ed and pest species not adapted to fire repopulate the area. Native wil dflowers can be replaced by low growing shrubs due to fire suppression (Taylor, 1998). Wildflower seed mixes used by restor ation specialists and homeowners often contain a variety of species specific to one habitat. This allows users to broadcast 15

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wildflower seed easily and effectively. Many wildflower handbooks stress the importance of selecting wildflower specie s of known provenance or ecotype, then planting the species within cl imates similar to ones they are adapted to (Miles, 1976). Taking advantage of provenance and ecotype oft en results in improved health and vigor of the plant. Genotypes from a local populati on are more likely to be tolerant of those stresses (Booth and Jones, 2001). O ne study found that Firewheel (Gaillardia pulchella [Asteraceae]) plants derived from a seed source from West Florida were intolerant of South Florida conditions (Hammond et al ., 2007). A similar response was seen when the species red clover ( Trifolium pratense [Fabaceae]), orchardgrass ( Dactylis glomerata [Poaceae]) and narrowleaf plantain (Plantago lanceolata [Plantaginaceae]) planted in test sites across Europe were found to have a home site advantage and a decrease in transplant performance with an increase in distance from the collection site (Joshi et al., 2001) Norcini et al. (2001) found that local seeds from lyreleaf sage ( Salvia lyrata [Lamiaceae]) had a higher survival percentage as compared to non local seed sources. Also, local seeds from tickseed ( Coreopsis lanceolata [Asteraceae]) and lyreleaf sage began flowering and reached full fl ower earlier as compared to non local ecotypes (Norcini et al., 2001). Another study comparing six wildflower species collected from outside vs. inside Florida found that thos e from outside the state adapted poorly to North Florida conditions, based on features su ch as length of flowering period, flower number, and disease incidence (Norcini et al., 1998). Harper-Lore and Wilson (2000) clarify that climat e and geological variation may be more important to plant establishment than geographical di stance. Elevation is another important consideration when planting. Lippit et al. (1994) recommend that seeds be planted in the same zone 16

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within 150 m of elevation. Even when a wildflower is native to an area, it should, like any other plant, be studied for the ideal soil type, moisture, and pH requirements to determine if it is appropriate for a planting site (Paulson, 1989). There are some issues associated with collecting plants from a local source. A limited collection area due to urban developm ent makes collection more difficult and time consuming. Collecting seed in the wild can be complicated because uneven ripening can exist at a single location (Pfaff et al., 2002). Landscape Potential of Two Florida Native Wildflowers There are currently 300-500 species of wildflowers commercially available, representing only 3-5% of the countrys native populati on (Milstein, 2005). October flower (Polygonella polygama (Vent.) Engelm. & A. Gray [Polygonaceae]) and sandhill wireweed ( Polygonella robusta (Small) G.L. Nesom & V.M. Bates [Polygonaceae]) are two Florida native wildflowers that have limit ed commercial availability. Many Florida wildflower handbooks neglect this genus entir ely, with a few offering only limited descriptions. However, Haehle and Brookwe ll (1999) note the aes thetic appeal of the genus, stating that Polygonella spp. would make a lovely addition to a wildflower garden. Both species have distinct characteri stics that differentia te them from other commercially available species, making them excellent options for homeowners and restoration specialists looking for unique native wildflowers (Table 1). P. polygama var. polygama is a perennial, widespread wildflower that is found in sandhills, flatwoods, and scrubs, in full to partial sun. The spatulate to linear light green leaves are approximately 0.5 to 2 cm (0.2 to 0.8 in) long and 0.25 cm (0.1 in) wide. The stem has some jointing visible every 2 to 3 cm (0.8 to 1.2 in). Its prolific, miniature, cream to yellow flowers usually appear in late fall on a terminal raceme 2 to 5 cm (0.8 to 17

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2 in) long, thus garnering it s common name October flower. Distribution includes the coastal plain regions of the southeastern United States (PLANTS Database, 2008). It is frequently found in open spaces with su rrounding flora such as scrub oaks ( Quercus spp. [Fagaceae]), gopher apple ( Licania michauxii [Chrysobalanaceae]) saw palmetto ( Serenoa repens [Arecaceae]) false rosemary ( Ceratiola ericoides [Empetraceae]), and pines (Pinus spp. [Pinaceae]) (Wunderlin and H ansen, 2003; Osorio, 2001; Taylor, 1998). Wunderlin and Hansen (2003) also describes a second, though less common variety, Polygonella polygama var. brachystachya (Meisn.) that is found mostly in flatwoods and has leav es 0.5-1.0 mm wide. A second Polygonella species, P. robusta, is more commonly found in open sand, full sun environments along the coasts of Florida (PLANTS Database, 2008). This mounding perennial has linear shaped leaves 3 to 5 cm (1.2 to 2 in) long and 0.25 cm (0.1 in) wide. The stems appear to be join ted due to a sheathing petiole, with fibrous hairs at each node. Like P. polygama, it is also floriferous, but with terminal pink to cream miniature flowers that appear sporadically throughout the year on racemes 3 to 10 cm (1.2 to 4 in) long. Though the species has only been found in Florida, it is well distributed throughout the state. Accompanying flora include: saw palmetto ( Serenoa repens [Arecaceae]), turkey oak ( Quercus laevis [Fagaceae]) gopher apple ( Licania michauxii [Chyrsobalanaceae]) prairie clover (Dalea spp. [Fabaceae]), yucca ( Yucca spp. [Agavaceae]), and some xerophytic grasses (PLANTS Database, 2008; Wunderlin and Hansen, 2003; Osorio, 2001; Taylor, 1998) Though neither species is widespread in cultivation, some native nurseries in Florida have attempted to grow P. polygama and 18

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P. robusta from seed. However, germination is often under 25% (N. Bissett, personal communication). The ornamental potential and limited propagation knowledge of P. polygama and P. robusta warrants further investigation. The overall objective of th is thesis project is to determine effective sexual and as exual propagation methods of P. polygama and P. robusta to increase their availability and us e in urban landscapes and restoration projects. Propagation by Seed Genetic diversity during sexual reproduction results from i ndependent assortment of chromosomes and crossing over during meiosis and fusion of gametes during fertilization. Plants grown from seed will theref ore be genetically diverse compared to plants from cuttings. Genetic diversity withi n populations for outplanting is a goal in many restoration projects. Using direct seeding methods for projects also eliminates the production process in the nursery. This may reduce labor costs and other expenses as maintenance of mature plants would be unnecessary. Germination is loosely defined as emergence of the radicle through the seed coat (Bradbeer, 1988). From a physi ological perspective, germination begins with water uptake by the seed and ends with the elonga tion of the embryonic axis (Bewley and Black, 1994). Unless dormant, seeds wi ll germinate if appropriate moisture, temperatures, and aerobic cond itions are sensed (Bradbeer, 1988; Bewley and Black, 1994). To germinate and establish readily, s eeds should have high viability and vigor. However, viability and vigor may be reduced when seeds ar e not collected and stored correctly. Seeds should be collected when the fr uit is mature, usually indicated by a change in color. After collection, seeds s hould be dried quickly and stored in a cool 19

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place at a moisture level low enough to prev ent premature germination, the formation of disease, and seed aging (Justice and Bass, 1978; Lippit et al., 1994). However, levels of desiccation and cold that are too low ma y damage seeds of some species (Justice and Bass, 1978). Dormancy is a seed trait that prevents ger mination when conditions are favorable for germination but the probability of seedling survival is low (Baskin and Baskin, 2001; Fenner and Thompson, 2005). Baskin and Ba skin (2001) recommend testing for dormancy by subjecting seeds to a range of simulated seasonal temperatures for a period of four weeks. Seeds are considered dormant if no or little germination occurs at the end of the four week period (Finch -Savage and Leubner-Metzger, 2006; Baskin and Baskin, 2001; and Bradbeer, 1988). When this test is extend ed for longer than four weeks it is possible that the seed would hav e broken dormancy, thus rendering the test results inconclusive (Baskin and Baskin, 2004a). Five types of dormancy are currently re cognized. Physical dormancy (PY) results from water impermeable seed or fruit coat s. Physiological dormancy (PD) can be characterized by the inability of embryos to rupture covering structures such as testa, endosperm, perisperm, and/or pericarp (Bradbeer, 1988; Baskin and Baskin, 2004a). Physiological dormancy may be caused by an embryo with a low growth potential, covering structures have low oxygen permeabi lity, or covering structures that are resistant to pressure exerted by elongati ng embryos change over time (Baskin and Baskin, 2001). Embryos that are undifferentiated or under developed at shedding are considered morphologically dormant (MD). Morpho-physiolog ical dormancy (MPD) is a combination of PD and MD and is commonl y found in wildflowers of temperate 20

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deciduous forests (Baskin and Baskin, 2004a). Finally, seeds with combinational dormancy (PY + PD) have impermeable coats and some physiological inhibition of the embryo (Baskin and Baskin, 2004a). Dormancy in native wildflowers may last anywhere from several weeks to many months, with some species possessing more than one type of dormancy mechanism (Bradbeer, 1988; Baskin and Baskin 2004a). Dormancy has been found to occur more commonly in s eeds of native plants than the seeds of cultivated plants (Paulson, 1989). There are several treatments that c an be applied to alleviate dormancy and promote germination. Scarificat ion to the testa or fruit tissues can improve imbibition and overcome PY. For the Florida native wildflower summer farewell ( Dalea pinnata [Fabaceae]), mechanical scarification was more successful in overcoming dormancy than acid scarification (Perez et al., 2009). Su lfuric acid scarification was successful however in increasing germinatio n in some woody plant species of Texas (Vora, 1989). Using warm and/or cold stratification, soaki ng the seeds in gibberellic acid (GA), or allowing the seeds to after-ri pen by storing in a cool and dry environment can alleviate PD. In two rare species of buckthorn ( Rhamnus spp. [Rosaceae]), germination was increased when the seeds collec ted in the summer received co ld stratification for at least one month (Sharma and Graves, 2005). For Leavenworths tickseed ( Coreopsis leavenworthii [Asteraceae]) physiological dormancy wa s broken by exposing the seed to specific temperature ranges, eit her cooler temperatures or cooler temperatures followed by warmer temperatures (Kabat et al., 2007) Elevated temperatures that alleviate dormancy in some species can be damaging in others, as seen in some species of Australian native forbs (Willis and Groves, 1991) In this same study, other species 21

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showed an increase in germinat ion when treated with GA, though it did not overcome any dormancy mechanisms (Willis and Groves, 1991). GA was helpful in overcoming the inhibitory effect of light on native Australian understory species (Bell et al., 1995). Using GA or stratification can also allow the embryos to further develop and overcome MD. It is suggested that MPD and PY + PD c an be overcome by one or more of the previously recommended treatment s (Baskin and Baskin, 2004a). Propagation by Cuttings Asexual, or vegetative, propagation may be used when growing plants from seed is impractical or not possible due to non viable or dormant seed (Ingram and Yeager, 1990). In some cases, plant s are propagated asexually to determine the sex of the finished plant. In hollies, female plants are desired for their red berries which are aesthetically pleasing and attr act wildlife. Producing plan ts using asexual propagation will result in a genetic clone of the parent plant. This method is commonly used in the horticulture industry to maintain the charac teristics desired in a popular ornamental. Asexual propagation may also be used to wi den production times that are usually restricted by seed availability (Guo et al., 2009). Finally, plants grown from cuttings often require less maintenance and time to produce a finished product (Phillips, 1985, Graham et al., 2006). Plant development is regulated by six ty pes of hormones: auxins, gibberellins, cytokinins, ethylene, abscisic acid, and bras sinosteroids (Graham et al., 2006). Auxins are plant hormones that play a role in many plant activities, including root initiation and development. Auxins accumulate in the epide rmal cells of root initials during the formation of lateral roots (Taiz and Zeiger, 2006), although the effects of auxin on root formation are not universal. The four major advantages of applying auxin to vegetatively 22

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propagated tissues are an increased rooting percent, reduced time to root initiation, increased quality of roots, and greater uniformity of the cutting (Macdonald, 1986). Once the roots are formed, the auxin concentrations are highest in the root tip, promoting the production of cells at the meristem (Taiz and Zeiger, 2006). Auxins typically interact with other plant hormones; in the case of cytokinins, the ratio determines t he initiation of root or shoot. Examples of naturally occurring auxins are indole-3-acetic acid (IAA) and 4chloroindole-3-acetic acid (4-Cl-IAA) (Taiz and Zeiger, 2006). Indole-3-butyric acid (IBA), previously believed to be synthetic, was also found to occur naturally in plants as a conversion product of IAA. The synthetic auxin 1-Naphthaleneacetic acid (NAA), in conjunction with IBA, has been shown to be mo re effective in promoting rooting than IAA. Root initiation of the Florida native beach sunflower ( Helianthus debilis ssp. debilis Flora Sun [Asteraceae]) improved when cuttings were treated with 2000 ppm of IBA (Norcini and Aldrich, 2000). Ho wever, regardless of the concentration the use of auxin did not improve the survival or growth of th e plant during container production (Norcini and Aldrich, 2000). Auxin application may also be deleterious in high concentrations, often causing damage to the tissue result ing in discoloration or death (Macdonald, 1986). Growth abnormalities such as leaf epinas ty, stem curvature, growth inhibition, and intensified leaf pigmentation may also occur with increasing auxin concentrations (Grossmann, 2000). It is also important to consider cutting material when choosing an auxin concentration. Stem cuttings can be taken at different stages of vegetative growth. Those taken from the current seasons grow th can be either softwood or semi-hardwood (Ingram and Yeager, 1990). Softwood cuttings are taken from succulent plant tissue that 23

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is from the new spring and summer gr owth (Ingram and Yeager, 1990). Softwood cuttings are commonly used in vegetative propagation because the tissue has yet to become lignified and the shoots are in an active state of growth (Macdonald, 1986). In contrast, semi-hardwood cuttings are of matured growth from the current seasons growth (Ingram and Yeager, 1990). Hardwood grow th is matured growth that is from previous seasons growth (Ingram and Yeager 1990). In a study on the Florida native rusty blackhaw ( Viburnum rufidulum [Adoxaceae]), cuttings fr om hardwood treated with 9000 ppm of IBA resulted in 100 % rooting while cutting from softwood treated with 6000 ppm of IBA reached 87% rooting (Griffin 2008), suggesting that hardwood cuttings often require higher concentrations of auxin as compared to softwood. Griffin (2008) also found that softwood cuttings that rece ived 0 ppm IBA still rooted minimally, while hardwood cuttings exhibited no rooting. It is recommended that cuttings of perennial plants should be taken in late spring as new growth is beginning to harden (Miles, 1976). They should also be collected before flowering. During floral initiation and anthesis a plant will reallocate resources towards production, development, and function of flowers instead of roots. ORourke (1940) observed that hardwood cuttings of blueberry ( Vaccinium atrococcum [Ericaceae]) with flower buds rooted poorly w hen compared to cuttings containing only vegetative buds. Cutting success may also be dictated by collection time after flowering. Guo et al. (2009) found that cu ttings taken from Peony ( Paeonia Yang Fei Chu Yu [Paeoniaceae]) at 70 days after flower had the highest rooting percentage when compared to 40 and 10 days after flower. Regar dless of cutting type, age, or location, many studies stress the import ance of collecting cuttings from healthy stock plants, as 24

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this has been directly correlated to rooting success (Guo et al., 2009; Druege et al., 2004; Thetford et al., 2001) After collection, cuttings are placed in moist media to facilitate growth of adventitious roots. Root init iation and survival of the cuttings can be augmented by the media. The media serves the dual function of keeping the cuttings moist while also maintaining good drainage. False rosemary ( Ceratiola ericoides [Empetraceae]), a species native to the Florida scrub and sandhills, showed minimal effect of root initiation when treated with IBA and NAA (Thetford et al., 2001). However, when comparing different media, it was found that the cuttings were most successfully rooted in a media with good drainage (Thetford et al., 2001), a resu lt not surprising for a species that naturally occurs in open sand. Thus m edia must be tailored to the moisture requirements of the species. In order for the plant to properly photosynthesize it r equires sunlight, oxygen, and water. Cuttings lack the root system required fo r the uptake of water from the soil, thus dessication of stem and leaf tissues must be avoided. Misting the cuttings helps manage the temperature of the cuttings and limit water loss by transpiration. However, cuttings that are kept too wet may be susceptible to fungal or bacterial pathogens (Miles, 1976). Ensuring proper water levels in the cuttings enables the plant to successfully undergo photosynthesis. Carbohy drates can then be utilized in the development of adventitious r oots. Another factor consider ed important by some is the proportion of endogenous nitroge n. Druege et al. (2004), f ound a positive correlation between the level of internal nitrogen and the amount of total sugars produced via photosynthesis for root production in geraniums ( Geranium spp. [Geraniaceae]). They 25

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found that cuttings with low le vels of nitrogen and carbon re sulted in greater leaf senescence (Druege et al., 2004). Experiment Preface This thesis will investigate methods of propagation of two native wildflowers that would make attractive additions to the horti culture industry. Chapter 2 will focus on the propagation of Polygonella polygama and Polygonella robusta by seed. Propagating native plants by seed is often the most desirable method to ensure genetic diversity. However, dormancy mechanisms often pr event optimal germination. Based on preliminary research on related species and discussions with nativ e plant growers, I hypothesize that seeds of both species w ill be dormant at shedding and after storage, thus justifying investigation of dormancy breaking treatments. Vegetative propagation offers an alternative when seeds are not viable, or the seeds are resistant to germination treat ments. In Chapter 3, propagation of P. polygama and P. robusta by stem cuttings will be discussed. I hypothesize that cuttings will show improved rooting when treated with a 2:1 co mbination of IBA and NAA. This is the recommended ratio for the optimal root ing of semi-hardwood cuttings. 26

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Figure 1-1. Differences in the morpholog ical characteristics and distribution of Polygonella polygama (left column) and Polygonella robusta (right column). Plant form and habit of P. polygama is more upright (A) versus the low growing P. robusta (B) Leaves of P. polygama are spatulate in shape (C), but linear for P. robusta (D). The flowers of P. polygama are yellow to creamy white (E) and pink to white on P. robusta (F). Plant distribution, noted by green color, is statewide for P. polygama and prevalent along the coast (G), where P. robusta has been found in fewer counties, but also frequently along the coast (H). Photo credits: A. Heather K. Muller, K. R uder, S. Wilson, and S. Woodmansee. Maps credits: www.florida.plantatlas.usf.edu. 27

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28 Figure 1-1. Continued.

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CHAPTER 2 GERMINATION AND DORMANCY STUDIES OF POLYGONELLA POLYGAMA AND POLYGONELLA ROBUSTA Introduction Seed dormancy, a phenomenon commonly asso ciated with native plants, delays germination of seeds when cond itions are not favorable for seedling establishment (Baskin and Bakin, 2004a; Paulson, 1989; Baskin and Baskin, 2001; Vleeshouwsers et al., 1995; Bewley and Black, 1994). While this benefits the seed and species by preventing wasted resources, it presents challenges to propagation efforts. Seed dormancy is classified into five cat egories (Baskin and Baskin, 2004b). Physically dormant (PY) seeds have water impermeable seed or fruit coats. Seeds with embryos that are undifferentiated or underdeveloped at shedding are considered morphologically dormant (MD). Seeds with physiological dorm ancy (PD) have covering structures that prevent the embryo from emergi ng. PD can also be attributed to low growth potential of the embryo, reduced oxygen permeability of the covering structures, changes in resistance to penetration of covering structures, or endogenous levels of plant hormones such as abscisic acid (ABA) and gibberellins (GA) (Bradbeer, 1988; Baskin and Baskin, 2004a; Baskin and Baskin, 2001). Morpho-physiological dormancy (MPD) is a combination of PD and MD, where the embryo is both underdeveloped and the covering structures provide resistance to further embryo development. MPD is commonly found in wildflowers of temperat e deciduous forests (Baskin and Baskin, 2004b). Combinational dormancy (PY + PD) refers to se eds that have impermeable coats and some physiological inhibition of the embryo (Baskin and Baskin, 2004b). With the exception of species within Coreopsis Rudbeckia, Gaillardia and Dalea (Perez et al., 2009; Rukuni, 2008; Kabat et al., 2007; Norcini and Aldrich, 2007; 29

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Danielson, 2005) dormancy and germination c haracteristics are largely unknown for Florida native wildflowers. Dormancy in wildflo wers can last from a few weeks to many months; and some species possess more than one type of dormancy (Bradbeer, 1988; Baskin and Baskin, 2004a). The presence of dormancy can be investigated by incubating freshly shed, mature seeds at vari ous simulated seasonal temperatures for 4 weeks, calculating the rate of water uptake, looking at seed and/or fruit coat anatomy and measuring embryo growth (Baskin and Baskin, 2004a). In addition to dormancy, improper seed storage can hinder native wildflower production. When seeds are kept in dry storage for long periods of time, viability may be reduced and the ability to germinate lost (Baskin and Baskin, 2001). Factors that are most important to successful seed storage are temperature, relative humidity, and storage time. Seeds can be dried naturally, us ing sunlight and wind, without significant loss of quality in the commercial production of some species (Black et al., 2006). However, prolonged hot and humid weather can a ccelerate seed deterioration (Priestly, 1986). These types of conditions are prevalen t in Florida and present a major challenge for seed storage within the state. If pr oper storage conditions are unknown, but wildflower germplasm must be stored, then the recommendation is that the sum of the storage temperature (oF) and relative humidity should be less than 100 (Norcini and Aldrich, 2007; Harrington, 1972) It should be noted that the duration of viability under these conditions is unknown for many wildf lower species and after-ripening may occur under certain storage conditions. After-ripening, which can occur in dry seeds of some species, is defined as the progressive loss of dormancy in dry mature seed (Black et al., 2006). This effect may 30

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terminate dormancy when oxygen conc entration and temperature increase and moisture levels decrease (Bewley and Black, 1994). This method is often used as a treatment to overcome physiological dorman cy; however in some species with physical dormancy, it can also be beneficial. Baski n and Baskin (2001) explain that as seeds after-ripen, the seed coat can dry and crack, thus becoming more permeable with time, and allowing water to enter and seeds to germinate more easily (Baskin and Baskin, 2001). Alternatively, seeds with higher water contents may lose viability or enter a secondary dormancy (Bewley and Black, 1994) In a study on several Florida native tickseed ( Coreopsis spp. [Asteraceae]), after-ripening effects were seen on all species, even with freshly harvested, less dormant se ed populations (Norcini and Aldrich, 2007). Lanceleaf tickseed ( Coreopsis lanceolata [Asteraceae]) dormancy was alleviated most effectively when the seeds were kept at 23% relative humidity and 17-19 oC (63-66 oF) (Norcini and Aldrich, 2007). In contrast maximum after-ripening of wild oat ( Avena fatua [Poaceae]) seeds was observed at around 40 oC (104 oF), and 10-12% relative humidity. Tang et al. (2009) found that dwarf rocket ( Olimarabidopsis pumila [Brassicaceae]) seeds dried to 5.6% moistu re content had the highest germination when incubated for 8 weeks at 30 oC (80 oF), and an additional 8 weeks at 4 oC. October flower ( Polygonella polygama (Vent.) Engelm. & A. Gray [Polygonaceae]) is a perennial wildflower with widespread distribution across Florida. It is commonly found in sandy scrub where it receives full to partial sun. The inflorescence consists of prolific, miniature, cream to yellow flowers. These flowers usually appear in mid to late fall, with seeds maturing and dropping in wi nter. In contrast, sandhill wireweed ( Polygonella robusta (Small) G.L. Nesom & V.M. Bates [Polygonaceae]) is more 31

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commonly found in open sand, full sun environm ents on the coasts of Florida. This mounding perennial is also very floriferous, but with terminal pink to cream colored miniature flowers covering each raceme. Fl owers appear sporadica lly throughout the year with fruit appearing soon after (PLANT S Database, 2008; W underlin and Hansen, 2003; Osorio, 2001; Taylor, 1998). T hese features make both native Polygonella species attractive candidates for the horti culture industry. Grower interest may be reduced however, if germination is delay ed due to seed dormancy. Developing an efficient, cost effective propagatio n plan for the seed production of Polygonella species may improve availability and increase use in the landscape. The objective of this study was to determine the extent of germination in P. polygama and P. robusta seeds. The following questions are addressed: 1) Are seeds dormant after harvest and subsequent storage; 2) What type(s) of dormancy ma y be present; and 3) How can dormancy be alleviated and germination promoted? Materials and Methods Seed Collection and Storage Achenes, henceforth referred to as seeds, of Polygonella polygama were collected from a native stand in Brooksville, Florida (28 51' 25''N, 82 25' 49''W) on February 15, 2008. This source is referred to as P. polygama (C), indicating the central collection site. Seeds were spread out in a single layer and exposed to ambient laboratory conditions (~ 24 C (75 oF), RH unknown) for 45 days before being cleaned of floral remains. They were then stored in a zip top plastic bag and placed again on the l aboratory bench top. Initial germination and viabilit y experiments commenced approxim ately 2 months later. An additional source of P. polygama seeds were collected from Pensacola, Florida (30 18' 48''N, 81 24' 39''W) on November 12, 2008. This source is referred to as P. 32

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polygama (N), indicating the northern collection si te. Seeds were spread out in a single layer and exposed to laboratory condition s as stated previously. Germination and viability experiments began approximatel y 5 months later. Seeds of Polygonella robusta were collected on April 10, 2008 in Hobe Sound, Florida (27o 01' 11''N 80o 06' 38''W). After collection, the seeds were cleaned, dr ied, and stored in plastic zip top bag in ambient laboratory conditions described above, for almost 4 months before undergoing testing. Pre-Germination Viability Assay Seed viability was examined using a tripheny ltetrazolium chloride (TZ) staining test before germination experiments commenced. Procedures were adapted from the Tetrazolium Testing Handbook (P eters, 2000). Four replicat ions of twenty-five seeds (n=100) each were nicked with a scalpel on the proximal end of the seed. Each replicate of nicked seeds was then placed into its own beaker containing 5 mL of 1% TZ solution (pH = 7). After 48 h of dark incubation at 35 oC (95 oF), the seeds were removed and triple rinsed with 15 mL of deionized wa ter. When data could not be immediately collected, the seeds were placed in 5 mL of deionized water and kept in the refrigerator at 5 oC for up to two weeks. Staining pattern s were viewed by bisecting the seed longitudinally and examining the endosperm and embryo under a dissecting microscope at 15 magnification. If embryos were la cking, unstained, or black, they were considered non-viable. If the entire embryo was stained a li ght pink, dark pink, or red, they were considered viable. Initial Germination Tests Four replications per treatment of 25-seeds (n = 800) of P. polygama (C) and P. robusta were surface sterilized in a 20% bleach solu tion for 10 min, followed by a triple 33

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rinse in deionized water. Seeds were placed in 32 Petri dishes (10.0 cm) on top of two sheets of blue blotter paper (8.9 cm diam eter Steel Blue Seed Germination Blotter, Anchor Paper Company, Minnesota, U.S.A. ) moistened with approximately 15 mL of deionized distilled water. Dishes were sealed with laboratory film. The seeds were then exposed to one of four simulated seas onal temperatures. T he 12 h alternating temperatures used were 22/11 C (72/52 F), 27/ 15 C (81/59 F), 29/19 C (84/66 F), and 33/24 oC (93/75 F). These temperatures repr esent the seasons of Florida during winter, early spring or late fall, early fall or late spring, and summer, respectively. For each treatment, four replicates were exposed to a 12 h daily photoperiod (80 mol m-2 s1, cool white fluorescent light), with the warmer temperature occurring during the lighted period. The remaining four replicates we re incubated in the dark by covering Petri dishes with two layers of aluminum foil. Treatments for initial germination were arranged in a 2 (illumination) 4 (temperature) fa ctorial. A randomized complete block design was used for this study. The experiment was carried out for 4 wk with germination counts taken once per week for the light tr eatment and at 4 wk for dark incubated seeds. After 4 wk, any remaining non-germinat ed seeds were assayed for viability as described previously with seeds that mo lded during the germination experiment counting as non viable seeds. Water Uptake by Intact and Scarified Seeds The extent to which imbibition occu rred was determined by measuring the increase in fresh mass on scarifi ed and non-scarified seeds. Seeds of P. polygama (C) and P. robusta were scarified by nicking the pericar p with a scalpel on the proximal end. The dry mass of four replic ations of 25-seed (n=100) was measured gravimetrically. After initial mass determinations (T0), seeds were placed into dishes containing two 34

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sheets of moistened blotter paper. After 0.25 h seeds were removed from the dishes, lightly blotted with a paper towel, and weigh ed to determine the fresh mass. Seeds were returned to their respective dishes, with the process repeating at 0.5, 0.75, 1-12, 24, and 48 h. Fresh mass increase was calculated using the formula [(Wi Wn)/ Wn] x 100, where Wi and Wn are the masses of imbibed and non-imbibed tissues (at T0), respectively. Seeds with obvious signs of fungal contamination or radicle emergence were noted and removed and mass determinat ion was adjusted accordingly. This experiment utilized a completely randomized design. Seed Anatomy and Morphology Seeds of P. polygama (C) and P. robusta were excised from their exocarps under a dissecting microscope using a scalpel, then nicked on the proximal and distal ends. Seeds that buckled when pressure was applied, thus identified as unfilled, and those with obvious signs of insect damage were ex cluded. Separated fruit coats (n = 10) and seeds (n = 10) were incubated for 24 h at 20 oC in plastic vials containing 1 mL of Trumps fixative. Following fixation, tissues were transferred to a buffer solution (0.1M cacodylate), microwaved at 180 watts for 45 se c with a vacuum at a pressure of 80 kPa, then allowed to sit for 15 min. After the buffer was removed, tissues were post-fixed in 20% osmium tetroxide for 24 h. Tissues were rinsed twice in buffer, then three times in deionized water. Samples were microwaved wit h a vacuum as before, then allowed to cool for 10-15 min between each rinse. T he seeds underwent dehydration using a graded EtOH series where the samples were held in each grade for 10-15 min, followed by two final steps in 100% acetone. Tissues were infiltrated with a graded Spurrs resin acetone series. Samples were heated in a micr owave set to 250 watts with a vacuum at 80 kPA for 3 min, with at l east one hour between each step. A fter the final infiltration 35

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step, seeds were removed and placed in mold s with fresh 100% Spurrs resin. The resin was allowed to polymerize in an oven set to 40 C for 48 h (Bozzola and Russell, 1999). Thick sections (ca. 680 nm) were made using a rotary microtome. Se ctions were placed on microscope slides and stained with Toluid ine Blue. The slides were then examined under a light microscope at 40 Images were captured using a digital camera (Olympus BH2-RFCA with a QImaging camera model Retiga 2000R FAST). Images were cropped and adjusted for color using QCapture Pro software (QImaging Cor poration, Surrey, British Columbia, Canada). Images of seed longit udinal sections, obtained from light microscopy studies, were used to calculate embryo : seed ratios. Images were uploaded into Adobe Photoshop (Adobe Systems Inc., San Jose, California, U SA) and the lengths of (n = 10) embryos and associated seeds were taken using the m easurement feature. Images of the fruit coat were examined to determine thickness based on the number of cells. Requirements for Warm or Cold Stratification The move-along experiment (Baskin and Baskin, 2003) was used to determine the extent to which seeds requi red warm or cold stratificati on. Four replications of 25seeds of P. polygama (C) and P. robusta were used in six temperature treatments (n=600). Seeds were surface sterilized and pl aced on blue blotter paper in Petri dishes. Treatments were adapted from Baskin and Baskin (2003) to mimic seasonal changes in Florida. The treatments consist ed of four control chambers set to 22/11, 27/15, 29/19, or 33/24 C; and two move-along treatments th at began with the summer temperature (33/24 C) or the winter temperature ( 22/11 C) (Table 2-1). Germination data was collected once per week for one year. 36

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Effect of Cold Stratification on Seeds Propagated in the Greenhouse Six replications of 1 seed (n=48) of P. polygama (N) were placed in plastic ziptop bags containing moistened vermiculite. The bags were then placed in a dark refrigerator at 5 oC (35 oF) for 0, 2, 4 or 8 wk. Seeds were then removed from the bags and half were surface sterilized using a 20% bleach solution for 10 min. Sterilized and non sterilized seeds were sown in trays c ontaining Fafard 2P. The trays were then placed in a misthouse where they received mi st for 8 sec every 10 min for the first week, and then 5 sec every 20 min for subsequent weeks. Treatments for cold stratification were arranged in a 2 (sterilization) 4 (stratification duration) factorial. A randomized complete block design was us ed for this study. Emergence data was collected once a week for six weeks. Effects of Gibberellic Acid on Seed s Propagated in the Laboratory and Greenhouse Four 25-seed replicates (n=500) of P. polygama (C) and P. robusta were placed on blue blotter papers moistened with gibberellic acid (GA3) solutions of 0, 1, 10, 100, or 1000 ppm for 24 h. After imbibition, seeds were surface sterilized with 20% bleach solution for 10 min. All seeds were then incubated at 22/11 oC (72/52 F) with a 12 h photoperiod. Germination data was collected once a week for four weeks. The effect of GA3 on germination was tested again in the greenhouse on seeds of P. polygama (N). Six replications of 1 seed (n= 48) were placed on blotters moistened with 0, 10, 100, or 1000ppm of GA3. After 24 h, half of the seeds were sterilized with 20% bleach solution for 10 min while the remain ing half were not sterilized additionally, then all sown in trays containing Fafard 2P. The trays were then placed in a misthouse where they received mist for 8 sec every 10 min for the first week and then 5 sec every 37

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20 min for subsequent weeks. Treatments were arranged in a 2 (sterilization) 4 (GA3 concentration) factorial. A randomized comple te block design was used for this study. Emergence data was collected once a week for six weeks. Initial Seedling Emergence and the E ffect of Different Propagation Media Additional germination tests were carried out in the greenhouse to test seedling emergence under simulated prod uction conditions. To test for emergence, seeds of P. polygama (N) were separated into sterilized and non sterilized treatments. Each treatment consisted of six r eplications of 6-seeds (n=72) Those that were sterilized were placed in a 20% bleach solution for 10 min. After sterilizat ion, both treatments were sown into 72cell trays filled wit h a peat based germination media (Fafard 2P, Conrad Fafard Inc., Massachusetts, U.S.A.). The trays were then placed in a misthouse where they received mist for 8 sec every 10 min for the first week and then 5 sec every 20 min for subsequent weeks. A randomized co mplete block design was used for this study. Emergence data was collected weekly for six weeks. Seeds (n=72) of P. polygama (N) were subjected to an additional experiment to assess the effects of media on germination. Th irty-six seeds were surface sterilized with 20% bleach solution for 10 min while the remaining half received no bleach (six replications of 6-seeds). Seeds were then sown on top of moistened media and lightly covered with a 0.5 cm (0.2 in) layer of the same media. T he medias used consisted of a peat based soilless mix (Fafard 2P, peat:per lite 3:2), fine quartz sand approximately 1mm in diameter (QUIKRETE Play Sand, Atlanta, Georgia, U.S.A), and a native mix. The native mix consisted of approximately 40% fine pine bark, 25% Fafard 2P, 25% sand, and 10% vermiculite. Fafard 2P was chosen because the composition is common to production media. Pure sand was used because both species are known to occur in 38

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areas with soil comprised lar gely of sand. Finally, the nat ive mix was used because it simulates the composition of media used by Florida native nurseries. The trays were then placed in a misthouse where they received mist for 8 sec every 10 min for the first week, and then 5 sec every 20 min for subsequent weeks. Treatments were arranged in a 2 (sterilization) 3 (media type) factor ial. A randomized complete block design was used for this study. Emergence data was collected weekly for six weeks. Data was analyzed using class comparisons for media treatments. Data Analysis For experiments carried out in the lab incubators, seeds were recorded as germinated when radicle emergence was at least 2 mm in length. For these experiments the final percent germination was adjusted by removing contaminated seeds from the calculation. However, seeds with fungal contaminat ion were considered non viable in the post germinat ion viability test. To determine the emergence percent for the seeds in the greenhouse, data was colle cted when the hypocotyl was visible above the soil line, usually more than 2 mm. Ge rmination rate was estimated for all experiments by determining the mean germinati on time (MGT). Data were transformed using the arcsine of the square root when t he range in percent germination was greater than 40 (Little and Hills, 1978). Non transfo rmed data are presen ted. Analysis of variance was performed using the PROC GLM procedure in SAS v. 9.1 (SAS Institute, Cary, North Carolina). Results Pre-Germination Viability Assay and Initial Germination Tests TZ staining indicated the initial viability of all sour ces to be greater than 50% (Table 2-2). All viable seeds of P. polygama (C) and P. robusta germinated to 100% 39

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when incubated at 22/11C. Moreover, germination ranged between 73 and 88% for viable seeds of either spec ies incubated at 29/19C (Table 2-3). However, the lowest germination (> 6%) occurred at 27/15 oC or 33/24 oC for viable P. polygama seeds. Germination was somewhat higher (~ 30-63%) for P. robusta seeds incubated at these temperatures. No germination was observed at any temperature co mbination for seeds of either species incubated in the dark (dat a not presented). However, according to the post-germination TZ test for seeds in complete darkness, less than 5% of P. polygama and 12% of P. robusta remained viable (Table 2-4). Germination rate is presented in Table 2-3 as mean germination time (MGT). Mean germination time for seeds of P. polygama at 27/15 and 29/19 oC was almost twice as rapid com pared to MGT at 22/11 oC. However, the MGTs were not significantly different (F2,6 = 1.77; p = 0.25). In seeds of P. robusta MGT was not significantly different between treatments (F2,6 = 1.11; p = 0.39). Water Uptake by Intact and Scarified Seeds Fresh mass increased for both species and scarification treatments, indicating uptake of water (Figure 2-2). Initially, P. polygama (C) seeds that were scarified showed the greatest increase in fresh mass; however by the end of the 48 h experiment the scarified seeds showed a 61% increase from their dry mass as compared to 74% increase in the seeds that were non-scari fied. This difference between scarification treatments was signif icantly different (F1,3 = 12.9; p = 0.04). P. robusta showed a similar pattern, where scarified seeds initially had a greater increase. In contrast to P. polygama at the end of the 48 h ex perimental period, the difference between scarified and non-scarified seeds of P. robusta was not significant (F1,3 = 2.59; p = 0.21). 40

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Seed Anatomy and Morphology The average length of P. polygama (C) embryos was 1.6 0.1 mm, with seeds averaging 1.7 0.1 mm. Embryos of P. robusta averaged 1.5 0.2 mm, with seeds averaging 1.6 0.2 mm (Figure 2-3A). Therefore, embryo : seed ratios were 0.95 0.01 and 0.94 0.01 for P. polygama (C) and P. robusta respectively. Moreover, fully developed embryos (i.e. cotyledons, embryo nic axis, and radicle) were clearly distinguishable in longitudinal sections (Figure 2-3A) Neither species seed nor fruit coats c ontained cells with lignified walls, nor palisade layers of macrosclerid s or osteosclerids (Figure 2-3B and 2-3C). Instead, the cell layers in the seed coats were observed to be one to two cells thick. Additional images of only the fruit coat show layers tw o to four cells thick (data not shown). Requirements for Warm or Cold Stratification After one year, seeds of P. polygama (C) incubated in control chambers showed reduced germination as compared to the move-along treatments (Figure 2-4). Seeds in the move-along chambers had a final germi nation of about 79% and 67% when started in summer and winter temperatures, respecti vely. For both move-along treatments, a dramatic increase in the slope of the germi nation was visible when the incubator was changed from warmer to cooler temperatures ( illustrated in Figure 2-4 by black arrows). The lowest germination perc entages occurred for seeds incubated at 29/19 and 33/24 oC, where germination reached 15% and 0%, respectively. Post-hoc mean separation showed that final germination percent at di fferent simulated seasonal temperatures were significantly different (F5,18 = 35.02; p < 0.01). Mean germination time was shortest for seeds in the 22/11 oC treatment (115 days) and longest for 27/15 oC (236 days). MGT for the move-along treat ments fell in the middle at 180 days for summer and 213 41

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days for winter. Post-hoc mean separation showed that all treatments were significantly different (F5,18 = 7.02; p < 0.01). Seeds of P. robusta in the move-along chambers had a final germination of 67% when begun in summer temperatures and 73% when begun in winter temperatures (Figure 2-4). Again both move-along treat ments had an increase in germination when seeds were moved from warmer to cooler te mperatures. However, the slope of the line for P. robusta was not as steep as was seen for P. polygama. In the control chambers, seeds performed the poorest when kept in 33/24 oC, reaching a final germination of less than 20%. A post-hoc mean separation showed that final germination percentage at simulated seasonal temperatures were significantly different (F5,18 =4.90; p = 0.005). Germination rates, as det ermined by MGT, were much different from P. polygama. All seeds that germinat ed in 22/11 and 33/24 oC did so in less than 20 days, whereas it took nearly 90 days for both move-along treatments. A post-hoc mean separation showed that mean germination times were significantly different from each other (F5,18 = 8.34; p < 0.001). Effect of Cold Stratification on Seeds Propagated in the Greenhouse Figure 2-5 displays the range of percent emergence for seeds of P. polygama (N) treated with 0, 2, 4, or 8 wk of cold (5C ) stratification. For non sterilized and sterilized treatments, emergence was greatest when seeds were stratified for 2 wk (39 and 31%, non-sterilized and sterilized, respectively). No emergence was observed for seeds treated in 8 wk of cold strati fication, regardless of steriliza tion. Class comparisons of the main effects of weeks in cold stratification were significant (F3,40 = 20.1; p < 0.01), but sterilization was not (F1,40 = 0.0; p = 0.89). The difference betw een 2 wk vs. 0 wk was significant (F7,40 = 7.08; p = 0.01), whereas 4 wk vs. 0 wk was not significant (F7,40 = 42

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0.23; p = 0.63). The interaction of stratifica tion sterilization was not significant (F3,40 = 2.5; p = 0.07). Trends observed for emergence percent found a linear decreasing trend when seeds were not sterilized (t2,19 = -3.6, p < 0.01) and a convex quadratic form when sterilized (t2,19 = -2.58, p = 0.02). Emergence rate, determined by MGT, was shortest for the control treatment (less than 10 days for both sterilization treatments) (Figure 2-5). A convex quadratic trend was observed in MGT for the sterilized treatment (t1,23 = -2.58, p = 0.02) while a negative linear trend was seen in the unsterilized treatment (t1,23 = -3.60, p < 0.01). Effects of Gibberellic Acid on Seed s Propagated in the Laboratory and Greenhouse P. polygama (C) seeds treated with 1000 ppm of GA3 reached a final germination of 33%, which was nearly double that of the control (16%) (F igure 2-6). When regressed, the trend in germi nation was a linear increase (t1,19 = 4.79, p < 0.01). There was no trend observed in MGT. GA3 concentrations of 100 and 1000 ppm had a positive effect on the germination percent of seeds of P. robusta (Figure 2-7). The trend seen in this data was a linear increase (t1,19 = 2.48, p = 0.02). There was no trend when observing MGT. For seeds of P. polygama (N) sown in the greenhouse (Figure 2-7) no trends were observed in GA3 concentration for sterilized. The highest germination percent (58%) occurred in seeds that were not sterilized and were treated with 10 ppm. The lowest percent germination (33%) occurred when se eds were not sterilized and treated with 100 ppm. The main effects of sterilization (F1,40 = 0.5; p = 0.48) and GA3 treatments were not significantly different (F2,40 = 0.2; p = 0.92). The interaction of sterilization treatment was also not significant (F3,40 = 1.69; p = 0.18). Mean germination time also 43

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had no observed trend due to GA3 concentration or sterilization treatment. Germination took 25 days when treat ed with 10 ppm of GA3 and no sterilization. The shortest period occurred when seeds were tr eated with 1000 ppm of GA3 and sterilized (14 days). Again, the main effect s of sterilization and GA3 treatments were not significantly different (F1,40 = 0.0; p = 0.85 and F2,40 = 0.2; p = 0.89, respectively). The interaction of sterilization x treatment wa s also not significant (F3,40 = 1.78; p = 0.17). Initial Seedling Emergence and the E ffect of Different Propagation Media In the greenhouse, unsterilized seeds of P. polygama (N) emerged less (17%) then sterilized seeds (28%). However, a post-hoc mean separation showed that the sterilization treatments were not significantly different (F1,11 = 1.74; p = 0.22). When comparing MGT, again the treatments we re not significantly different (F1,10 = 0.54; p = 0.48). Sterilized seeds of P. polygama (N) responded with the greatest percent emergence (~81%) when sown on sand (Figur e 2-1). As seen in Table 2-5, the sterilization treatments did not significantly affect emergence percent, whereas media did (F2,30 = 33.8; p < 0.01). Class comparisons of the main effects of treatment show that the difference between native mix vs. Fafard 2P was not significant (F1,29 = 0.04; p = 1.00), whereas sand vs. Fa fard 2P was significant (F1,29 = 29.68; p < 0.01) (Table 2-5). The mean germination time was fastest fo r seeds sown on sand (16 days when not sterilized). Again, the MGTs of native mix vs. Fafard 2P was not significant (F1,29= 0.00; p = 0.96) whereas significant differences in MGT occurred for sand vs. Fafard 2P (F1,29 = 6.29; p = 0.02). 44

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Discussion Physical dormancy does not occur in seeds of P. polygama nor P. robusta because both intact and scarified seeds imbibed water at similar rates and to similar final fresh mass percetages. Seeds with PY do not regularly imbibe water (Perez et al., 2009; Jayasuriya et al., 2007; Baskin et al ., 2006b, Turner et al., 2005, Baskin and Baskin, 2001). Although the final increase in fresh mass was statistically different for scarified vs. non scarified seeds of P. polygama the difference is not large enough to be considered biologically significant as bot h seeds regularly imbibed water. On the contrary, scarification of six physically dormant genera of Rhamanceae showed that seeds treated first in hot water had a steady increase or an initial dramatic increase in fresh mass whereas non treated increased only minimally (Turner et al., 2005). Regular increases in fresh mass were also observed for seeds of P. robusta. These patterns of fresh mass increase for both scarification tr eatments and species are similar to what was observed in the non-physically dormant seeds of woollyjoint prickly pear ( Opuntia tomentosa [Cactaceae]) when placed on a wet substrate (Orozco-Segovia et al., 2007). Additionally, both Polygonella species lacked the anatomy typically observed in species reported to possess PY. Light microscopy images of the physically dormant whitestar ( Ipomoea lacunosa [Convolvulaceae]) show the presence of long sclereid cells perpendicular to the seed coat (Jayasuriya et al., 2007). Moreover, two species of Stylobasium spp. (Surianaceae) displayed thickene d outer layer of palisade cells (Baskin et al., 2006b). Likewise, morphological dormancy is not present for either Polygonella species. For example the embryo : seed ratio was close to 1 indicating fully mature embryos that did not require additional time to dev elop. The embryo : seed ratio for the 45

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morphologically dormant bishops weed ( Aegopodium podagraria [Apiaceae]) was 0.14 (Vandelook et al., 2009). The images of the Polygonella species were similar to morphologically dormant seeds of Haskap ( Lonicera caeulea var. emphyllocalyx [Caprifoliaceae]) that were a llowed to mature after 3 weeks in incubation to promote embryo growh (Phartyal, 2009). Despite the lack of PY and MD, dormancy has long been known to occur in some members of the Polygonaceae (Ransom, 1935). Vi ability and initial germination studies indicate some form of dorman cy is present in seeds of both P. polygama and P. robusta after harvest and subsequent storage For example, over 20% of P. polygama collected in north Florida remained unstained during init ial viability testing (Table 2-2), but these embryos appeared intact, suggesting that a por tion of the seed population was dormant (Baskin et al., 2006a; Norcini et al., 2006; Peters, 2000). Furthermore, all temperature treatments exce pt for 22/11 oC, had seeds that remai ned ungerminated that stained pink or red in the post-germination TZ te st. These stained seeds were considered dormant and reached 33% when P. polygama was kept in 33/24 oC (Table 2-3). The occurrence of dormancy is also supported by the initially low germination occurring at each initial incubation temper ature (Table 2-3). An initia l germination test on both species found that emergence pe rcent reached nearly 86% for P. polygama and 68% for P. robusta when held in a 20/10 oC incubator for 16 wk (S. Wilson, personal communication). Though seeds did not germi nate in when subjected to the dark treatment, it is not believed that dormancy pr evented the germination. Interestingly, both species, regardless of temperature, had lo w viability when subject ed to a post TZ test (Table 2-4). This low viability may indicate that seeds will not survive in their natural 46

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seed bank. Since scrub and sandhill ecosystems are known to have a low germinable seed bank, future germination studies using buried seed should be carried out on these species. Seeds of both species were exposed to various simulated seasonal temperatures in a move-along study. Sudden increases in germination were observed when temperatures were changed from warmer to cooler (33/24 to 29/19 to 27/15 oC). Final germination of the seeds kept in t he cooler incubators (27/15 and 22/11 oC) was not as high as the move-along treatments. The increase in germination as temperature decreases, indicates that seeds require a period of warm temperatures followed by cool for maximum germination. Since final germination percent was not as high for the winter control as it was for both move-along treatm ents, this indicates that the succession of fall to winter conditions maximizes germination. This trend was seen in Hawaiian seeds of Hamakua clermontia (Clermontia pyrularia [Campanulaceae]) where 12 wk at 25/15 oC followed by 20/10 oC resulted in 90% germination (Baskin et al., 2005). Dormant seeds of Virginia blue bells ( Mertensia virginiana [Boraginaceae]) also responded with greater germination in cooler temperatures, reaching 91% when incubated at 5 oC as compared to 0% in 25/15 oC (Baskin and Baskin, 2003). Seeds receiving 2 weeks of cold stratification and sown in the greenhouse reached a final germination greater than the control. The improvement of germination using cold stratification was also seen on initially dormant wild buckwheat ( Eriogonum [Polygonaceae]) seeds (Meyer and Paulsen, 2000). The same pattern was observed in seeds of wild buckwheat ( Polygonum convolvulus [Polygonaceae]), where stratification at low temperatures of 2 or 10 oC was believed to increase embryo growth potential 47

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thus overcoming dormancy and promoti ng germination (Metzger, 1992). The common name of P. polygama is October flower, referencing its flower time. Seeds of P. polygama are usually shed in fall when temperatures are still relatively warm in Florida, meaning that seeds are initially exposed to relatively warm temperatures then low temperatures as the seasons progress supporting the germination patterns observed during the move-along experiment. Though P. robusta flowers more sporadically throughout the year, a common ti me for the seeds to reach the shedding stage is late summer/early fall (A. Heather personal observation). Gibberellic acid is used to improve germination in seeds with physiological dormancy. GA3 is thought to increase the activity of cell wall degrading enzymes allowing the radicle to emerge, induce the synthesis of ot her enzymes that transport nutrients necessary for embryonic growth, a nd act as an antagonist to ABA, which has been shown to play a significant role in the maintenance of physiological dormancy (Baskin and Baskin, 2001; Nicolas et al., 1996). Germination of Polygonella seeds also improved following t he application of GA3, suggesting that physiological dormancy was alleviated and germination enhanced with this tr eatment. For example, final germination was almost two times greater in seeds of both species when tr eated with 1000 ppm of GA3 compared to controls (Figure 2-6 and 2-7). This finding was similar to what was seen in beech ( Fagus sylvatica [Fagaceae]) seeds where dormancy was overcome by a long period in cold temperatur es or the application of 100 M GA3 (Nicolas et al., 1996). Once their physical dormancy was broken, germination in some physiologically dormant seeds of sumac species ( Rhus spp. [Anacardiaceae]) treated with 500 or 1000 mg/L GA3 was more than 5 times greater than the control (Li et al., 1999). Unlike what was 48

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observed in the controlled setting of the incubators, percent germination was not positively correlated with GA3 concentration when applied on seeds grown in the greenhouse. This inconsistency may be explai ned by the very warm temperatures that occurred in the greenhouse (daytime highs were often > 35 oC) versus the cooler temperatures used in the incubator s (set to reach a maximum of 22 oC). Using the classification system sugges ted by Baskin and Baskin (2004b) and the results discussed above, it is suggested that seeds of P. polygama and P. robusta exhibit non-deep physiological dormancy af ter harvest and s ubsequent storage. Characteristics of non-deep PD include promot ion of germination when GA is applied, dormancy break occurring with the use of warm or cool stratification, and after-ripening occurring during storage (discussed below). Seeds exhibiting non-deep PD may require as little as 5 days at cold stratification to promote germination (Baskin and Baskin, 2001). Seeds of P. polygama and P. robusta responded to 2 weeks of 5 oC when sown in the greenhouse. Sumac species ( Rhus spp. [Anacardiaceae]) exhibiting non-deep PD showed improved germination when cold stratifi ed 7, 15, or 25 days (Li et al., 1999). Baskin and Baskin (2004b) go on to delineate between five types of non-deep PD. It is concluded that Polygonella species in this study fall under type 2 non-deep physiological dormancy. As seeds move from dormancy to non-dormancy, species that display type 2 will germinate as temperatures move from high to low. The obvious issue that persists for this st udy is the factor of storage. Seeds were collected and then kept in the lab (approximately 25 oC or 77 oF) for 2-5 months prior to experimentation. Though other members of Polygo naceae have had substantial success in maintaining viability in storage (Japanese knotweed [ Fallopia japonica ]) 49

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(Forman and Kesseli, 2003), it is unknown if P. polygama or P. robusta responded as well. If germination experiments had begun imm ediately after collection, results may have been dramatically different as seeds are typically the most deeply dormant at the shedding stage (Baskin and Baskin, 2001; Bewley and Black, 1994). However, it is possible that seeds of the Polygonella species studied here experienced the loss of dormancy over time due to the process of after-ripening (Foley, 2001) As after-ripening proceeds, seeds become more germinable over a wider range of conditions (Bewley and Black, 1994). This includes the ability to germinate more readily for a greater number of temperatures, and a greater sensitivity to chemical treatments such as GAs (Foley, 2001). Sometimes storage (i.e. after-ripening) is used as a treatment to increase germination when dormancy mechanisms are unknown. Seeds of marigold ( Calendula spp. [Asteraceae]) germinated at higher rates after being stored for multiple years in a cool, dry environment (Widrlechner, 2007). Aside from the possible types of dormancy present in Polygonella seeds and how to alleviate these and promot e germination, the experiments reported here investigated the role of propagation media in germinat ion. Though the experiment utilized only 72 seeds and was carried out once, it was c oncluded that sand was the most effective germination media (Figure 21). This study can be suppor ted by a germination study carried out in a greenhouse in Fort Pierce on Fafard germination media. There, seeds (n = 100) emerged to only 4% for P. polygama in May and 25% for P. robusta in December (S. Wilson, personal communication). This is also ecologically supportable as both species are found to naturally occur on areas previously disturbed and consisting of open sand (S. Woodmansee, per sonal communication). Both species are 50

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51 also found in areas commonly associated with fire, indicating that organic matter found in the topsoil would be consumed in t he burn. However, using only sand as a germination media in the nursery setting is impractical due to its high bulk density and low water holding capacity. Instead, it would be recommended to use a media with high drainage and low organic matter. Several general conclusions can be drawn from these studies. First, the results support that both species possess non-deep physiological dormancy after harvest and subsequent storage. Non-deep physiologica l dormancy in these species may be alleviated most effectively by cold stratification at 5 oC for 2 weeks or the application of warmer, summer like temperatures follow ed by cooler, winter like temperatures. Second, dormancy may also be alleviated by periods of indoor storage. However, it must be stressed that the effects of storage temper ature, storage relative humidity, and storage duration on possible after-ripening or seed viability of Polygonella species remains unknown at this time. Finally, in t he nursery setting, seeds will have the highest rate and percent germination when sown on sand based media.

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Table 2-1. Change in temperatures detailed in the move-along experiment with number of weeks seeds spent in incubation at each temperature. Weeks at temperature Move-Along treatments Control treatments 12 22/11 oC Winter 33/24 oC Summer 22/11 oC 27/15 oC 29/19 oC 33/24 oC 4 27/15 oC Early Spring 29/19 oC Early Fall 22/11 oC 27/15 oC 29/19 oC 33/24 oC 4 29/19 oC Late Spring 27/15 oC Late Fall 22/11 oC 27/15 oC 29/19 oC 33/24 oC 12 33/24 oC Summer 22/11 oC Winter 22/11 oC 27/15 oC 29/19 oC 33/24 oC 4 29/19 oC Early Fall 27/15 oC Early Spring 22/11 oC 27/15 oC 29/19 oC 33/24 oC 4 27/15 oC Late Fall 29/19 oC Late Spring 22/11 oC 27/15 oC 29/19 oC 33/24 oC 12 22/11 oC Winter 33/24 oC Summer 22/11 oC 27/15 oC 29/19 oC 33/24 oC 52

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Table 2-2. Pre-germination viability of a ll seed sources tested using 1% TZ solution. Total viability determined by adding total number of seeds stained red and pink. Data is presented as m ean percent standard error. Tetrazolium test (%) Species Red Pink White Black Empty Total viable (%) P. polygama (C) 65.5 4.7 11.9 2.6 1.0 1.0 21.6 6.6 0.0 0.0 77.4 6.3 P. polygama (N) 66.7 3.6 11.5 5.9 21.8 5.1 0.0 0.0 0.0 0.0 78.1 5.1 P. robusta 47.3 6.7 6.2 1.1 3.0 1.0 8.2 3.9 35.3 5.9 53.5 6.8 Table 2-3. Germination and TZ viability testing of Polygonella polygama (C) and Polygonella robusta seeds. Dormant (%) determi ned by total viable-final germination. Total viable via prege rmination TZ found by dividing Final germination by Total viable. Mean percentages and standard errors are shown. Species and temperature (oC) Final germination (%) Dormant (%) Total viable (%) Total viable via pregerminati on TZ (%) Mean germination time (d) P. polygama 22/11 30.1 5.3 0.0 0.0 30. 1 5.3 100.0 0.0 14.4 1.8 27/15 1.4 1.4 20.0 6.4 21.4 6.3 6.3 6.3 7.0 7.0 29/19 17.2 3.0 5.8 5.8 23.0 8.1 87.5 12.5 12.3 1.8 33/24 0.0 0.0 33.5 12.2 33.5 12.2 0.0 0.0 LSD(0.05) 9.6 23. 0 24.1 16.1 4.3 P. robusta 22/11 59.4 17.6 0.0 0. 0 59.4 17.6 100.0 0.0 9.3 1.3 27/15 55.9 9.2 32.1 5.7 88.0 7.4 62.6 7.9 9.6 0.4 29/19 26.1 11.5 17.1 10.2 43.2 20.1 73.3 16.3 8.2 1.2 33/24 8.3 8.3 22.2 15. 7 30.6 17.8 30.0 30.0 7.0 7.0 LSD (0.05) 27.9 24.4 32.0 3.4 2.4 53

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Table 2-4. Post-germination viab ility from the dark treatment at all temperatures tested using a 1% TZ solution. Total viability determined by adding total number of seeds stained red and pink. Data is presented as mean per cent standard error. Species and temperature (oC) Red Pink White Black Empty Total viable (%) P. polygama 22/11 1.0 1.0 0.0 0.0 31.0 4.4 3.0 1.0 17.0 1.0 1.0 1.0 27/15 3.0 1.9 1.0 1.0 36.0 15.1 1.0 1.0 8.0 4.3 4.0 2.3 29/19 1.0 1.0 2.0 2.0 15.0 3.4 4.0 1.6 12.0 4.0 3.0 3.0 33/24 0.0 0.0 0.0 0.0 42.0 11.8 4.0 4.0 14.0 7.4 0.0 0.0 P. robusta 22/11 0.0 0.0 0.0 0.0 7.0 3.4 2.0 2.0 7.0 7.0 0.0 0.0 27/15 6.0 3.8 0.0 0.0 8.0 4.3 4.0 2.8 7.0 4.7 6.0 3.8 29/19 10.0 4.2 1.0 1.0 4.0 2.8 3.0 1.0 13.0 4.4 11.0 5.0 33/24 4.0 1.6 1.0 1.0 3.0 1.9 4.0 1.6 12.0 1.6 5.0 2.5 Table 2-5. Analysis of variance table of emergence percent and rate for class comparison between sterilizat ion and media treatments of P. polygama (N). Source df Emergence MS Emergence p -value MGT MS MGT p-value Model 5 4406.4 <0.001 417.6 0.14 Sterilization 1 31.0 0.75 1.1 0.88 Media 2 10040.0 <0.001 199.6 0.02 Native mix v. Fafard2P 1 0.0 1.00 0.1 0.96 Sand v. Fafard2P 1 15060.1 <0.001 6.3 0.02 Sterilization x Media 2 960.6 0.05 12.0 0.77 54

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Media Soil Soil Mix Emergence (%) 0 25 50 75 100 Media Soil Mix Sand Mean germination time (days) 0 10 20 30 Figure 2-1. Emergence percent as an effect of media types and su rface sterilization on seeds of P. polygama (N). The native mix consisted of 40% fine pine bark, 25% Fafard 2P, 25% sand, and 10% vermi culite. Circles and columns in dark gray were non-sterilized treatments, while open bars denote sterilized treatments. Error bars represent the standard error of the mean. 55

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Time ( hours ) 0102030405 0 Fresh Mass Increase (%) 0 25 50 75 100 0 25 50 75 100 Polygonella polygama Polygonella robusta Figure 2-2. Increase in fresh mass of scari fied (solid circles) or non-scarified (open circles) seeds of P. polygama (top), or P. robusta (bottom). Error bars represent standard er ror of the mean. 56

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Figure 2-3. Thick sections of Polygonella seeds. (A) Longitudinal section of P. polygama seed showing fully developed embryo (em) with cotyledons (c) and radicle (r) surrounded by the endosperm (en). Magnification = 20. (B) The fruit coat (fc), remaining re sin layer (re), seed coat (sc) and endosperm (en) of P. polygama. Magnification = 40. (C) Close up of the endosperm (en), seed coat (sc) and resin layer (re) of P. polygama Magnification = t 100. (C). Black bars represent 0.1 mm. 57

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58 Time (weeks) 16111621263136414651 Ger mion (%) inat0 25 50 75 100 Polygonella polygama 22/11 o C 27/15 o C 29/19 o C 33/24 o C Summer Winter Time (weeks) Polygonella robusta 0 25 50 100 75 16111621263136414651 Figure 2-4. Cumulative germination of seeds collected in central Florida of P. polygama (top) and P. robusta (bottom) incubated at simu lated seasonal temperatures. The key shows the four control temperatures and the two move-along treatments beginning at 33/24 oC for summer and 22/11 oC for winter. Arrows indicate when move-along treatments changed temperatures.

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2D Graph 9Time in cold stratification (weeks) 0248 Mean germination time (days) 0 10 20 30 Emergence (%) 0 25 50 75 100 Figure 2-5. Final emergence percent (t op line graph) and mean germination time (bottom bar graph) of P. polygama (N) seeds after moist stratification in the dark at 5 oC for 0, 2, 4, or 8 wk. Circles and columns in dark gray were treatments not ster ilized, while white was ster ilized. Error bars represent standard error of the mean. 59

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Germination (%) 0 25 50 75 100 GA3 (ppm) 0 1 10 100 1000 Mean germination time (days) 0 10 20 30 Figure 2-6. Final germination percent (top line graph) and mean germination time (bottom bar graph) of P. polygama (C) seeds after soaking for 24 h in varying concentrations of GA3 and subsequent incu bation at the constant temperature of 22/11 oC (72/52 oF) for 28 days. 60

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Germination (%) 0 25 50 75 100 GA3 (ppm) 0 1 10 1001000 Mean germination time (days) 0 5 10 15 20 Figure 2-7. Final germination percent (top line graph) and mean germination time (bottom bar graph) of P. robusta seeds after soaking for 24 h in varying concentrations of GA3 and subsequent incu bation at the constant temperature of 22/11 oC (72/52 oF) for 28 days. 61

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62 Emergence (%) 0 25 50 75 100 GA3 (ppm) 0 10 100 1000 Mean germination time (days) 0 10 20 30 Figure 2-8. Final emergence percent (t op line graph) and mean germination time (bottom bar graph) of P. polygama (N) seeds after soaking for 24 h in varying concentrations of GA3 and subsequent growth in the misthouse for 42 days. Circles and columns in dark gray were treatments not sterilized, while white was sterilized. Error bars repres ent standard error of the mean.

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CHAPTER 3 EFFECT OF AUXIN APPLICATION ON ROOTING OF SOFTWOOD CUTTINGS OF POLYGONELLA POLYGAMA AND POLYGONELLA ROBUSTA Introduction Even when seeds are collected at the appropriate time, properly stored, and subjected to favorable conditions, dorm ancy mechanisms often exist that delay germination (Baskin and Baskin, 2004a; Finch-Savage and Leubner-Metzger, 2006; Bradbeer, 1988). Though both October flower ( Polygonella polygama (Vent.) Engelm. & A. Gray [Polygonaceae]) and sandhill wireweed ( Polygonella robusta (Small) G.L. Nesom & V.M. Bates [Polygonaceae]) flower prolifically, propagation by seed can be difficult due to physiological dormancy that is characterized by delayed, erratic, or reduced germination (Heather et al., 2009) In addition, seed collection of Polygonella spp. from natural populations can be rest ricted by varying seasonal conditions, management practices, or narrow harvest windows (Ingram and Yeager, 1990). As an alternative to producing natives by seed, vegetative propagation can often improve efficiency, enhance product uniformity, generate finished plants quicker and with greater reliability. Vegetative propagation, or asexual propagation, is the production of a new plant from a cutting of a parent plant. For this research projec t, stem cuttings were taken from plants in wild populations and then root ed in media trays kept in a misthouse. Auxins are plant growth regulating substances that ar e frequently applied to the basal end of fresh cuttings to induce the form ation of adventitious roots. For instance, rooting of softwood cuttings of Indian rosewood ( Dalbergia sissoo [Fabaceae]) was enhanced when 100 mg/L of either indole-3-butyr ic acid (IBA) or 1-naphthaleneacetic acid (NAA) was applied (Puri and Verma, 1996) Auxins such as IBA and NAA are 63

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commonly used in the propagatio n industry. For example, the commercially popular liquid rooting product known as Dip N Grow (DipN Grow Inc., Clackamas, OR) is a combination of auxins at a ratio of tw o parts IBA to one part NAA. Though this combination is often used in the industry, most researchers utilize the chemicals independently to optimize species specif ic rooting responses. Both IBA and NAA elicited similar results in percent rooti ng and mortality when applie d to Fraser fir ( Abies fraseri [Pinaceae]), though NAA produced a greater number of and longer roots (Rosier et al., 2004). In one experiment on sugar maple ( Acer saccharum [Sapindaceae], combinations of IBA and NAA were used in a 1:1 ratio, though this ratio did not produce longer or a greater percentage of roots as compared to IBA or NAA alone (Alsup and Cole, 2004). Vegetative propagation has served as a reliable alternative for several other native coastal or scrub species including false rosemary ( Ceratiola ericoides [Empetraceae]) (Thetford et al., 2001), dune sunflower ( Helianthus debilis [Asteraceae]) (Norcini and Aldrich, 2000), and for the mi cropropagation of sea oats ( Uniola paniculata [Poaceae]) (Valero-Aracama et al., 2007). Thetfo rd et al. (2001) recommend IBA and NAA concentrations below 5000 ppm for rooting false rosemary. Norcini and Aldrich (2000) found 2000 ppm IBA was optimal to induce ad ventitious roots on dune sunflower. To our knowledge, vegetativ e propagation protocols have not been developed for Polygonella species. In unpublish ed preliminary data of P. polygama using only IBA, M. Thetford (personal communica tion) found that the control cuttings rooted at 83% and increased to 86-98% with 1000 to 5000 ppm of IBA. Additionally, P. robusta cuttings exposed to 1000 to 5000 ppm K-IBA produced more roots than those treated with 0 64

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ppm K-IBA. In these prelim inary studies, rooting substrate (Fafard 3P mix or perlite:vermiculite) did not a ffect rooting (M. Thetford, personal communication). The overall objective of this study was to determine if effective methods could be developed to root P. polygama and P. robusta cuttings collected from different sites in Florida. Specific objectives were to (1) evaluate the effectiveness of various auxin concentrations and combinations on rooti ng, and (2) determine if collection site influences rooting success. Materials and Methods Cutting source and auxin treatments Natural populations of P. polygama and P. robusta were identified and the habitats they were growing in were characteri zed by assessing neigh boring species, burn history, population number, population health, disturbance affinity, and distribution (Table 3-1). P. polygama is often found along the edge of scrub and mesic habitats (Table 3-2). P. robusta is found growing in full sun on sandhills. Both species are commonly associated with highly dist urbed areas (S. Woodmansee, personal communication). Cuttings of P. polygama and P. robusta were collected from locations in central or south Florida during the summer of 2008 and 2009 (Table 3-1, Table 3-2). Terminal (i.e. softwood) stem cu ttings, approximately 10 cm (3.9 in) in length, were collected in the morning from approximately 35 plants of each species. Pruning shears were surface sterilized with 95% ethanol in between species. All cuttings were immediately placed between moist paper towels in zip top plastic bags. Plastic bags were then wrapped with newspapers and stored bet ween icepacks in a foam cooler for transportation to the processing site in Gaine sville. The cuttings were stored inside the cooler for up to 24 h prior to sticking. 65

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Prior to treatment, cuttings were tri mmed to 11.5 cm (4.5 in) and the foliage removed from the basal 3 cm (1.2 in) of each cutting. Nine auxin treatments were formulated from 10,000 ppm st ock solutions of indole-3-butyric acid (K-IBA) and 1naphthaleneacetic acid (K-NAA) that were diluted to appropriate concentrations with distilled water. The basal 1 cm (0.4 in) of each cutting was quick dipped for 8 sec in one of nine IBA:NAA solutions (0:0, 0:250, 0:500, 500:0, 500:250, 500:500, 1000:0, 1000:250, 1000:500 ppm) and allowed to air dry pr ior to sticking (T able 3-3). Cuttings were inserted 2 cm (0.8 in) deep into 72-plug cell trays filled with pre-moistened soilless Fafard 2P media (Fafard Inc., Apopka, FL). The trays were placed in a mist house where intermittent mist operated 8 sec every 10 min during the daytime. After 2 weeks, mist was reduced to 5 sec every 20 mi n. Greenhouse temperat ure was set at 27 oC (80 oF) with a natural photoperiod of 14 h and an approximate photon flux of 300 molm-2s1 at the propagation bench. Data Collection Cuttings were removed from mist after 6 weeks ( P. robusta ) or 8 weeks ( P. polygama ). Media was carefully removed from roots by rinsing with tap water. For each experiment, rooting was assessed using four parameters: a visual root quality rating (rooting index), rooting per centage, root length, and root number. A cutting was considered rooted if it had one adventitious root 0.5 cm in length. Rooting index was based on a scale from 1 to 5, where 1= dead, 2= alive without roots, 3= light rooting that does not hold media, 4= medium rooting that holds media that is mostly removed with a light shake, and 5= heavy rooting that holds onto media that must be removed with washing. Rooting percentage was calculated using the number of cuttings that received 66

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a rating of 3, 4, or 5. R oot length was recorded as the longest primary root from each cutting. Experimental Design and Data Analysis For each species, a split plot experiment al design was used with the collection site as the main plot and the auxin treatments as the subplot. The subplots were arranged in a randomized complete block design with 6 cu ttings per treatment (n=270). Each auxin treatment was replicated 5 ti mes. Root percent data was transformed by taking the arcsine of the square root. However, untr ansformed data are presented. Data were subjected to ANOVA and trend analysis using SAS v.9.1 (SAS Institute, Cary, North Carolina). Significance of main effect s and interactions was determined using SAS PROC MIXED. Trend analysis was carri ed out using SAS PROC GLM. Results Polygonella polygama At 6 weeks, the P. polygama cuttings did not produce sufficient roots as compared to what had been observed in P. robusta at 6 weeks, therefor e the experiment was extended for an additional 2 weeks. At 8 weeks, regardless of collection site, no single auxin treatment produced roots that were consistently best among all measured response variables (Table 3-4) Average root index is shown in Figure 3-2 and Figure 33. The root index across both sites and all auxin combinations was somewhat low and did not exceed a rating of 2.8. Root index reached 2.6 0.2 and 2.5 0.1 when cuttings from the central Florid a source were treated wit h 500:0 and 0:500 (IBA:NAA), respectively. For the southern source of P. polygama, the greatest root index of 2.8 0.4 and 2.8 0.2 was achieved when cu ttings were treated with 500:0 and 1000:500 IBA:NAA, respectively. Rooting percentage ranged from 27-63% for both collection sites 67

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of P. polygama The greatest rooting for the centra l collection source of 60 11.3 % was reached when cuttings were treated wit h 500:0 IBA:NAA. For cuttings collected from the southern source, r ooting ranged from 40-63% with the greatest rooting of 63.3 9.7 % receiving the treat ment of 1000:250 IBA:NAA. Neither source produced more than 5 root s per cutting. The greatest number of roots (4.2) was reached when cuttings from south Florida were treated with 500:0 IBA:NAA. The least number of roots per cutting (2.3) wa s obtained when the central collection was treated with 500:250 IBA:NAA. Number of root s for cuttings collected in south Florida ranged from 3.8 to 5.1. The greatest number of roots of 5.1 0.7 was reached when treated with 1000:500 I BA:NAA. Finally, root l ength varied from about 2 to 5 cm. The longest root length, 5.2 0. 3 cm, was reached when cuttings from central Florida were treated with 1000:0 IBA:NAA. The shortest root length was also found in the central source for cutti ngs in the control group. Both rooting percent and root i ndex for the central source of P. polygama had the greatest response when treated with IBA at 500:0. Root i ndex, percent and number all responded the least to IBA:N AA at 500:250. For root l ength, the 1000:0 and 1000:500 IBA:NAA treatments produc ed roots that were more than t wice the length of the control plants. Cuttings taken from the south Florida source had similar rooting success compared to cuttings from the central Florid a source (Table 3-4). For example, root index reached 2.8 at 500:0 and 1000:500 IBA:NAA. Root i ndex, percent and number were lowest when treated with 0:250. As seen in Table 3-4, only one interaction was significant for P. polygama IBA NAA for root index. When IBA was held cons tant at 0 ppm, a concave quadratic trend 68

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was observed. In comparison, a convex qua dratic curve and an incr easing linear trend were seen when NAA was held constant at 0 and 250 ppm, respectively (Table 3-6). Polygonella robusta Regardless of collection site, no single auxin treatment produced roots that were consistently best among all measured traits (Table 3-5). Averages of each root index treatment are depicted in Figure 3-4 and Figure 3-5. The r oot index ranged from 1.6 to 3.6. The cuttings collected from the central source had the greatest root index when treated with 1000:0 IBA:NAA (3.3 0.3). Thos e collected in south Florida reached a maximum root index of 3.6 0.3 when treated wit h 500:250 IBA:NAA. Percent rooting was highly variable, rangi ng from 10-80%. Less variati on among treatments occurred from the central source, with greatest percent rooti ng (76.7 11.3%) at 1000:0 IBA:NAA. When cuttings collected in south Fl orida were treated wit h 500:250 IBA:NAA, rooting percent reached 80.0 6.2%. The number of roots per cutting for P. robusta ranged from 3 to nearly 11. Both central and south Florida cuttings produced the greatest number of roots when treated with 1000:250 IBA:NAA (9.6 1.7 and 10.9 2.0, respectively).The range for root length varied greatly from about 2 to 10 cm. The greatest r oot length occurred with the 1000:250 IBA:NAA treatment for central Florida cuttings (8 .3 1.0 cm) or 0:250 IBA:NAA for south Florida cuttings (10.1 0.3 cm). Considering all rooting factors, cuttings from the cent ral source performed best when treated with 1000:0 IBA:NAA. The central source had the lowest rooting for all factors when treated with 0:500 IBA:NAA. Cutt ings from the south source performed best when treated with 500:250 IBA:NAA. Lowes t percent rooting and root index were found when south Florida cuttings we re treated with 0:500 IBA:NAA. 69

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As seen in Table 3-5, some interactions are significant for P. robusta For the central source, there is a significant li near decrease in rooting index when IBA concentration was zero and NAA concentration increased. Alternatively, a significant linear increase in rooting index occurred when IBA concentration increased but NAA remained at 500ppm. However, the rooting inde x for cuttings from south Florida can be explained using a quadratic function when IB A concentrations were held constant but NAA concentration increased. In these inst ances the shape of the quadratic curves were concave when IBA was held constant at 0 ppm and convex for 500 and 1000 ppm. A quadratic trend, convex in shape, was observed when NAA was held constant at 250 ppm and IBA increased (Table 3-7). For the in teraction of site IBA NAA, similar trends were seen for percent rooting as was seen for root index (Table 3-8). An increasing linear trend was visible for cutti ngs collected in central Florida when NAA was held constant at 250 ppm and IBA in creased. When IBA was held constant and NAA increased, percent root ing for the southern source could all be described by a quadratic curve. When IBA was held at 0 ppm the shape can be described as concave, whereas when IBA is held at 500 and 1000 ppm the shape is convex. When NAA is held constant at 0 ppm, the percent rooting is shaped as a quadratic concave curve. Alternately, NAA held at 250 ppm yiel ds a quadratic convex curve. When looking at root number the interaction of IBA NAA was significant. Only one trend was visible over increasing IBA concentrations, and one trend for increasing NAA concentrations (Table 3-9). When IBA was held constant at 1000 ppm a quadratic curve, convex in shape, was evident with increasing NAA concentrations. In contrast, holding NAA constant at 250 ppm, created an increasing linear trend with increasing 70

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IBA concentrations. Finally, trends in root l ength were evident by Site IBA and Site NAA interactions (Table 3-10). When Site IBA was pooled over NAA the central site had an increasing linear trend when IBA concentra tions were increasing. In contrast, for Site NAA pooled over IBA, a convex qua dratic curve was observed with increasing NAA concentrations. Discussion Results for both species revealed no single auxin treatment to be consistently most effective in promoting the formation of adventitious roots. However, for both species collected from both locations, im proved rooting was often seen when IBA was greater than 0 ppm, regardle ss of NAA concentration. Overall, cuttings taken from P. robusta had a greater response to auxin application, with larger root index results and percent rooting than P. polygama. Rooting hormones have been shown to have variable e ffects based on species. In a study on fifteen taxa of snowbells ( Styrax spp. [Styracaceae]), it wa s found that rooting of 9 species was influenced by the application of I BA, five of which were positive (Griffin and Lasseigne, 2005). The im proved rooting of P. robusta may be explained in part by the condition of the plants at the collection site The southern site was characterized by a recently cleared scrub habitat with surroundi ng pines, oaks, and saw palmettos. The plants collected there appeared healthier with greater rates of new, green growth and thicker stems. Another potential influence in rooting success may be explained by collection time. The initial collection was done in late su mmer of 2008 due to environmental setbacks such as drought and fire. This meant that th e cuttings were collected just a few months prior to flowering. Many of the south P. polygama cuttings flowered during the 8 weeks 71

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in the mist house, while cuttings from south P. robusta flowered soon after data collection in the greenhouse (less than 10 weeks a fter collecting). This suggests that the cuttings may have allocated carbohydrates to flowering instead of rooting. This translocation of energy has been known as early as 1940 (ORourke) when hardwood cuttings of blueberry ( Vaccinium atrococcum [Ericaceae]) with flower buds did not root as well as those with vegetative buds. Though leve ls of rain and fire make it difficult to collect only softwood cuttings, higher concen trations of rooting hormones may be required to induce adventitious rooting based on time of collection. Sharma and Aier (1989) found that when semi-hardwood plum cuttings were collected in the summer they needed only 2000 ppm of I BA to reach optimal rooting. This concentration had to be increased to 3000 ppm when cuttings were taken in the fall or when dormant. Finally, hormone concentration has been know n to improve rooting success. Often when rooting trends were descri bed linearly, this indicates that using even higher levels of auxins might produce greater rooting. Only when the results were explained quadratically can it be assumed that the optimal auxin concentration was used. For cuttings of P. robusta collected in south Fl orida, both root index and percent rooting were described quadratically for IBA conc entrations (Table 3-7 and Table 3-8). However, the lack of significance for P. polygama may indicate that a greater number of auxin treatments with a greater number of r eplications at higher concentrations, should have been used (Table 3-5). It is also possible that other factors, such as misthouse conditions and propagation media may have contributed to variable results. The misthouse used had very low light and over head misters that frequently got clogged, resulting in occasional inconsistent wa tering. The media used here was only a peat 72

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based soilless media. In previous propagation work done on P. polygama also using a perlite/vermiculite media with K-IBA concentrations at 1000 and 2000 ppm and higher light levels, a significant interaction was obser ved between propagatio n media and root length (M. Thetford, personal co mmunication). Increase in rooting at higher light levels would be expected as bot h species grow well in full sun. Future experiments on these species shou ld be carried out to test other factors that are important to the success of root formation. Cutting ma turity, substrate, moisture, auxin concentration, year, and stock plant management can significantly affect root potential (Blythe et al., 2007; T hetford et al., 2001). These experiments only utilized tip cuttings collected in the summer. Thetford (2008) found that rooting percentage, rate, and number of roots did not di ffer with Fafard or perlite/ve rmiculite substrates. Since both Polygonella species are native to sandy sites, better drainage may improve rooting. The auxin concentrations used in this experiment were also fairly low. In order to make appropriate recommendations, additional experiments should be conducted with higher concentrations of both N AA and IBA individually and combined. 73

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Table 3-1. Collection date, site locati on, and site information for cuttings of Polygonella polygama (October flower) and Polygonella robusta (sandhill wireweed). Species Collection date Site name City Location Ecosystem type GPS coordinates Polygonella polygama 8/28/2008 Haney Creek Preserve Stuart South scrub 27 13' 35''N, 80 15' 16''W Polygonella polygama 6/9/2009 Withlacoochee State Forest Brooksville Central scrub/sandhill 28 51' 25''N, 82 25' 49''W Polygonella robusta 7/21/2008 Jonathan Dickinson State Park Hobe Sound South scrub 27o 01' 11''N 80o 06' 38''W Polygonella robusta 6/9/2009 Pine Ridge proposed landfill Orlando Central sandhill 28 o 29 32N 81 o 3 27W Table 3-2. Collection site c haracteristics for cuttings of Polygonella polygama (October flower) and Polygonella robusta (sandhill wireweed). Observations were noted by the co-collector, S. Woodmansee (personal communication). Species Location Dominating species Fire influence General observations Polygonella polygama South Pinus clausa, scrub oaks ( Quercus spp.), Serenoa repens, Ceratiola ericoides Fire suppressed, though recent wildfire ~100 individuals, little to no exotic plant influence, foot trail nearby Polygonella polygama Central Andropogon spp., Quercus geminata, Pinus clausa, Pityopsis, Aristida spp. Fire suppressed in scrub, sandhill burned recently ~300 individuals, plants declining slightly, though expected to improve with increased rain and fire Polygonella robusta South PInus clausa, scrub oaks ( Quercus spp.) Serenoa repens, Ceratiola ericoides Regular fire regime ~200 individuals occur in recently cleared area, healthy Polygonella robusta Central Pinus palustris, Serenoa repens, Quercus geminata, Selaginella spp., Liatris tenuifolia Fire suppressed ~400 individuals, disturbed area to become landfill in a few years, plants slightly smaller 74

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75 Table 3-3. Cuttings of October flower ( Polygonella polygama ) and sandhill wireweed ( Polygonella robusta ) were quick dipped in one of the following indole-3butyric acid (IBA) 1-naphthalen eacetic acid (NAA) used. IBA Concentration (ppm) NAA Concentration (ppm) 0 250 500 0 0 0 0 250 0 500 500 500 0 500 250 500 500 1000 1000 0 1000 250 1000 500

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Table 3-4. Effects of K-IBA and K-NAA treatments on rooting softwood cuttings of October flower ( Polygonella polygama ) collected from central (top) or south (bottom) Florida populations. Data presented as means standard error. Site NS NS NAA NS NS NS NS Site NAA NS NS NS NS IBA NS NS NS NS Site IBA NS NS NS NS IBA NAA NS NS NS Site IBA NAA NS NS NS NS Site Treatment Root index (scale 1-5) Rooting (%) Root number Root length (cm) Central 0 IBA, 0 NAA 2.0 0.1 30.0 6.2 3.3 1.1 2.2 0.3 Central 500 IBA, 0 NAA 2.6 0.2 60.0 11.3 3.6 0.2 4.0 1.0 Central 1000 IBA, 0 NAA 2.2 0.1 43.3 8.5 4.2 0.3 5.2 0.3 Central 0 IBA, 250 NAA 1.9 0.2 36.7 9.7 2.6 0.5 4.6 0.8 Central 500 IBA, 250 NAA 1.8 0.2 26.7 11.3 2.3 0.1 4.2 1.6 Central 1000 IBA, 250 2.4 0.3 46.7 16.2 3.6 0.5 4.4 0.3 Central 0 IBA, 500 NAA 2.5 0.1 56.7 4.1 3.2 0.4 3.8 0.6 Central 500 IBA, 500 NAA 2.3 0.3 50.0 15.8 3.3 1.8 3.0 0.5 Central 1000 IBA, 500 2.1 0.4 43.3 17.2 2.8 0.4 4.9 0.8 South 0 IBA, 0 NAA 2.7 0.3 53.3 9.7 3.8 0.7 2.6 0.6 South 500 IBA, 0 NAA 2.8 0.4 60.0 13.5 4.0 0.7 3.7 0.5 South 1000 IBA, 0 NAA 2.2 0.1 40.0 6.7 4.1 1.0 2.7 0.5 South 0 IBA, 250 NAA 1.8 0.2 26.7 11.3 4.0 0.8 4.8 0.7 South 500 IBA, 250 NAA 2.6 0.3 50.0 13.9 4.1 0.8 2.7 0.4 South 1000 IBA, 250 2.5 0.2 63.3 9.7 4.2 0.2 4.5 0.5 South 0 IBA, 500 NAA 2.6 0.3 50.0 9.1 4.1 0.7 2.8 0.6 South 500 IBA, 500 NAA 2.4 0.2 56.7 8.5 4.6 0.8 3.4 0.7 South 1000 IBA, 500 NA A 2.8 0.2 60.0 12.5 5.1 0.7 2.9 0.5 Non-significant (NS) at =0.05 (*), 0.01 (**), or 0.001 (***). 76

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Table 3-5. Effects of K-IBA and K-NAA treatments on rooting softwood cuttings of Sandhill wireweed ( Polygonella robusta ) collected from cent ral (top) or south (bottom) Florida populations. Data presented as means standard error. Site Treatment Root index (scale 1-5) Rooting (%) Root number Root length (cm) Central 0 IBA, 0 NAA 3.0 0.5 56.7 19.4 8.9 1.7 6.3 1.7 Central 500 IBA, 0 NAA 3.0 0.3 60.0 12.5 7.7 0.9 7.3 1.7 Central 1000 IBA, 0 NAA 3.3 0.3 76.7 11.3 7.6 1.0 7.9 1.4 Central 0 IBA, 250 NAA 2.9 0.2 66.7 5.3 6.2 0.7 6.1 2.2 Central 500 IBA, 250 2.8 0.4 60.0 15.5 7.5 1.6 8.2 1.9 Central 1000 IBA, 250 2.8 0.6 53.3 17.8 9.6 1.7 8.3 1.0 Central 0 IBA, 500 NAA 1.6 0.4 20.0 12.3 5.8 2.4 2.9 0.7 Central 500 IBA, 500 2.7 0.5 53.3 13.3 7.7 0.8 6.1 0.8 Central 1000 IBA, 500 2.9 0.2 63.3 9.7 7.3 0.4 8.1 1.0 South 0 IBA, 0 NAA 2.8 0.3 50.0 11.8 7.4 1.7 2.3 .1 South 500 IBA, 0 NAA 2.1 0.2 13.3 8.2 3.0 0.0 1.7 0.8 South 1000 IBA, 0 NAA 2.1 0.1 16.7 5.3 4.1 0.5 4.4 0.6 South 0 IBA, 250 NAA 2.1 0.1 10.0 6.7 5.0 0.0 10.1 0.3 South 500 IBA, 250 3.6 0.3 80.0 6.2 8.5 1.4 6.9 0.8 South 1000 IBA, 250 2.9 0.4 50.0 14.9 10.9 2.0 4.8 0.5 South 0 IBA, 500 NAA 2.9 0.2 53.3 6.2 6.4 1.2 6.8 1.9 South 500 IBA, 500 2.4 0.4 23.3 16.3 9.4 4.3 7.6 0.2 South 1000 IBA, 500 NA A 2.3 0.2 23.3 11.3 9.2 1.6 4.2 1.3 Site NS *** NS NAA NS NS NS Site NAA NS IBA NS NS NS NS Site IBA NS NS NS IBA NAA NS NS Site IBA NAA ** *** NS NS Non-significant (NS) at =0.05 (*), 0.01 (**), or 0.001 (***). 77

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Table 3-6. Trend analysis of P. polygama root index. Main effects equaled the source locations. Sub-plots equaled factorial combinations of IBA and NAA. ANOVA indicated that the interaction of IBA NAA was significant. NAA IBA 0z 250 500 L Q 0 2.33 1.85 2.58 NS ** 500 2.70 2.17 2.37 NS NS 1000 2.20 2.47 2.43 NS NS L NS NS Q NS NS zNon-significant (NS) or significant at =0.05 (*), 0.01 (**), or 0.001 (***). Table 3-7. Trend analysis of P. robusta root index. Main effects equaled the source locations. Sub-plots equaled factorial combinations of IBA and NAA. ANOVA indicated that the site IBA NA A interactions were significant. NAAz IBAz 0 250 500 L Q Central 0 3.00 2.93 1.60 NS 500 2.97 2.83 2.67 NS NS 1000 3.30 2.77 2.93 NS NS L NS NS Q NS NS NS South 0 2.83 2.07 2.90 NS ** 500 2.07 3.57 2.43 NS ** 1000 2.13 2.90 2.27 NS L NS NS NS Q NS ** NS zNon-significant (NS) or significant at =0.05 (*), 0.01 (**), or 0.001 (***). Table 3-8. Trend analysis of P. robusta percent rooting. Main effects equaled the source locations. Sub-plots equaled factorial combinations of IBA and NAA. ANOVA indicated that the site IBA NA A interactions were significant. NAAz IBAz 0z 250 500 L Q Central 0 52.06 55.02 20.59 NS NS 500 51.13 54.00 47.23 NS NS 1000 67.18 47.23 53.36 NS NS L NS NS Q NS NS NS South 0 45.00 11.87 46.95 NS ** 500 14.11 66.26 20.23 NS *** 1000 21.51 47.87 22.82 NS L NS NS NS Q *** NS zNon-significant (NS) or significant at =0.05 (*), 0.01 (**), or 0.001 (***). 78

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Table 3-9. Trend analysis of P. robusta root number. Main effe cts equaled the source locations. Sub-plots equaled factorial combinations of IBA and NAA. ANOVA indicated that the interaction of IBA NAA was significant. NAA IBA 0z 250 500 L Q 0 8.18 5.59 6.08 NS NS 500 5.34 8.00 8.53 NS NS 1000 5.83 10.27 8.27 NS L NS ** NS Q NS NS NS zNon-significant (NS) or significant at =0.05 (*), 0.01 (**), or 0.001 (***). Table 3-10. Trend analysis of P. robusta root length. Main effe cts equaled the source locations. Sub-plots equaled factorial combinations of IBA and NAA. ANOVA indicated that the Site IBA pooled ov er NAA (top) and Site NAA pooled over IBA (bottom) interactions were significant. Site IBA Central South 0 5.11 6.38 500 7.19 5.37 1000 8.08 4.45 L NS Q NS NS NAA Centralz South 0 7.16 2.80 250 7.53 7.23 500 5.69 6.17 L NS NS Q NS zNon-significant (NS) or significant at =0.05 (*), 0.01 (**), or 0.001 (***). 79

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80 Figure 3-1. Representation of 5 root index categories, from left to right, where 5= heavy rooting, 4= medium rooti ng, 3= light rooting, 2= alive with no roots and 1=dead. Cuttings are from P. polygama collected in south Florida

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Figure 3-2. Representative central Florida P. polygama cuttings 8 wk after being quick dipped in various rooting hormone concent rations. Starting at the top row, from left to right, NAA:IBA ppm, 0:0, 0:500, 0:1000, 250:0, 250:500, 250:1000, 500:0, 500:500, and 500: 1000. Scale bars represent 4 cm (1.6 in). 81

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Figure 3-3. Representative south Florida P. polygama cuttings 8 wk after being quick dipped in various rooting hormone conc entrations. Starting at the top row, from left to right, NAA:IBA ppm, 0:0, 0:500, 0:1000, 250:0, 250:500, 250:1000, 500:0, 500:500, and 500: 1000. Scale bars represent 4 cm (1.6 in). 82

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Figure 3-4. Representative central Florida P. robusta cuttings 6 wk after being quick dipped in various rooting hormone conc entrations. Starting at the top row, from left to right, NAA:IBA ppm 0:0, 0:500, 0:1000, 250:0, 250:500, 250:1000, 500:0, 500:500, and 500: 1000. Scale bars represent 4 cm (1.6 in). 83

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84 Figure 3-5. Representative south Florida P. robusta cuttings 6 wk after being quick dipped in various rooting hormone concent rations. Starting at the top row, from left to right, NAA:IBA ppm, 0:0, 0:500, 0:1000, 250:0, 250:500, 250:1000, 500:0, 500:500, and 500: 1000. Scale bars represent 4 cm (1.6 in).

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CHAPTER 4 CONCLUSIONS The intent of this thesis was to find effective methods of propagating October flower (Polygonella polygama (Vent.) Engelm. & A. Gray [Polygonaceae]) and sandhill wireweed ( Polygonella robusta (Small) G.L. Nesom & V.M. Bates [Polygonaceae]) to enable commercial production for use in landscape and restoration. Both species have tremendous value aesthetically and would provide ecological benefits to the natural landscape. The first method investigated was sexual, or seed propagation (C hapter 2). Seeds were tested for viability using a TZ solu tion and germinated in four alternating temperature regimes to mimic seasons in Florida. From these experiments it was determined that seeds of both species are fair ly viable, but also dormant. To determine the type of dormancy, seeds were subjected to various germination experiments. By monitoring fresh weight gain over time it wa s determined that intact or scarified seeds imbibed water regularly. Moreover, using light microscopy, seeds and fruit coats were examined for the presence of anatomy typical of tissues that do not imbibe water; for example, multiple lignified ce ll layers, palisade layers of thick-walled sclerids, and water gaps. None of these features were found. Therefore, it was determined that neither species possessed physical dormancy. By measuring the embryo to seed ratios of histological sections it was found that both species hav e fully developed embryos, and are therefore not morpholog ically dormant. Physiological dormancy was confirmed using a move-along experiment, where germi nation was highest when first treated with warmer temperatures, then mov ed to cooler ones. Additionally, gibberellic acid, known 85

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to improve germination in seeds with physiol ogical dormancy, showed a linear increase in germination with the increase in GA3 concentration for both species. To reflect more practical conditions found in a nursery setting (as opposed to germination in petri dishes placed in controlled incubators), P. polygama seeds were sown on various substrates and placed in a closed greenhouse with overhead mist (Chapter 2). Greatest germinatio n was achieved with sand as the substrate rather than the standard commercial mix or even the Fl orida native mix. Physiological dormancy was alleviated in some seeds of the populati on when seeds were moist stratified for two weeks prior to sowing. Contrary to the labor atory experiments, the application of GA did not improve germination. In each of the greenhouse experim ents, seeds were sterilized with bleach or not sterilized prior to sowing; however, sterilization had no effect on percent emergence. In Chapter 3 asexual propagation methods were investigated. Softwood cuttings were collected from natural populations in south and central Florida. Cuttings were subjected to nine auxin treatm ents and placed in overhead mist for 6-8 weeks. Visual root quality, root initiation, root length, and root number were assessed. Regardless of species, no auxin treatment proved to be most effective in promoting adventitious root formation. P. robusta cuttings appeared to have a greater response to auxin, with greater root indices an d rooting percentages, than P. polygama It was observed that the location from which cuttings were collect ed also affected root initiation and quality. Germination rates were found to increase wit h cold stratification and use of sand while rooting of cuttings was variable pot entially due to auxin concentrations, cutting type, media, and light. Therefore, it is conc luded that due to the results of this thesis 86

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87 these species can be successfully propagated fr om seed. However, more research is needed on the germination biology of these species; especially with seeds collected at the shedding stage. Furthermore, landscape trials including both species, transplanted from seedlings and cuttings, are currently bei ng held. Preliminary observations indicate that plants grown from s eed appear healthier and with better form then those grown from cuttings.

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LIST OF REFERENCES Alsup, C.M., and J.C. Cole. 2004. Stem cutting from Caddo sugar maple trees differ in their rooting potential. ISHS Acta Hort iculturae: XXVI Intl. Hort. Congr. 630:263269. Association for Florida Na tive Nurseries (AFNN). 2009. About Us 15 July 2009. Baskin, C.C. and J.M. Baskin. 2001. Seeds : Ecology, Biogeography, and Evolution of Dormancy and Germination. Acadademic Press, San Diego, C.A. Baskin, C.C. and J.M. Baskin. 2003. When br eaking seed dormancy is a problem, try a move along experiment. Na tive Plants 4:17-21. Baskin, C.C. and J.M. Baskin. 2004b. Germinat ing seeds of wildflowers, an ecological perspective. HortTechnol. 14:467-473. Baskin, C.C., Baskin, J.M., and Yoshinaga, A. 2005. Morphophysiological dormancy in seeds of six emdemic lobeliod shrubs (Campanulaceae) from the montane zone in Hawaii. Can. J. of Bot. 83:1630-1637. Baskin, C.C., Thompson, K., and J.M. Baski n. 2006a. Mistakes in germination ecology and how to avoid them. Seed Sci. Res. 16:165-168. Baskin, J.M. and C.C. Baskin. 2004a. A cla ssification system for seed dormancy. Seed Sci. Res. 14:1-16. Baskin, J.M., Baskin, C.C ., and K.W. Dixon. 2006b. Physica l dormancy in the endemic Australian genus Styloasium, a first report for the fa mily Surianaceae (Fabales). Seed Sci. Res. 16:229-232. Bell, D.T., Rokich, D.P., McChesney, C. J., and J.A. Plummer. 1995. Effects of temperature, light, and gibberellic acid on germination of seeds of 43 species native to Western Australia J. of Veg. 6:797-806. Bewley, J.D. and M. Black. 1994. Seeds: Physiology of Development and Germination, 2nd ed. Plenum Press, New York, N.Y. Black, M., Bewley, J.D., and P. Halmer (eds.) 2006. The encycolopedia of seeds: science, technology and uses. CABI Publishing, Cambridge, M.A. Booth, T.D. and T.A. Jones. 2001. Plants for ecological restoration: a foundation and a philosophy for the future. Na tive Plants J. 2:13-19. Bozzola, J.J. and L.D. Russell. 1999. Electr on micrscopy: principles and techniques for biologists 2nd ed. Jonesand Bartlett Publishers, Sudbury, M.A. 88

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Bradbeer, J.W. 1988. Seed Dormancy and Germi nation. Chapman and Hall, New York, N.Y. Christman, S.P. and W.S. J udd. 1990. Notes on plants endem ic to the Florida scrub. Fla. Sci. 53:52-73. Danielson, H.E. 2005. Production and performance of Gaillardia cultivars and ecotypes. MS Thesis. Dept. of Environ. Hor t., Univ. of Fla., Gainesville. Druege, U., Zerche, S. and R. Kadner 2004. Nitrogenand storage-affected carbohydrate partitioni ng in high-light-adapted Pelargonium cuttings in relation to survival and adventitious r oot formation under low ligh t. Ann. of Bot. 94:831-842. Fenner, M. and K. Thompson. 2005. The Ecol ogy of Seeds. Cambridge Univ. Press, New York, N.Y. Finch-Savage, W.E. and G. Leubner-Metzger. 2006. Seed dormancy and the control of germination. New Phytol. 171:501-523. Florida Native Plant Society (FNPS). 2003. Defi nition of a Florida Native Plant. 5 May 2009 Florida Wildflower Foundation. 2009. Introduction. 30 September 2009 Foley, M.E. 2001. Seed dormancy: an update on terminology, physiological genetics, and quantitative trait loci regulating germinability. Weed Sci. 49: 305-317. Forman, J. and R.V. Kesseli. 2003. Sexual reproduction in the invasive species of Fallopia japonica (Polygonaceae). Amer. J. of Bot. 90:586-592. Frances, A.L. 2008. Establishment and managem ent of native wildfl owers on Florida roadsides and former pastures. PhD Dissertation. Dept. of Environ. Hort., Univ. of Fla., Gainesville. Graham, L.E., Graham, J.M., and L. W. Wilcox. 2006. Plant Biol ogy. Pearson Education, Inc., Upper Saddle River, N.J. Griffin, J.J. 2008. IBA formulation, concentra tion, and stock plant growth stage affect rooting of stem cuttings of Viburnum rufidulum J. of Environ. Hort. 26:1-3. Griffin, J.J. and F.T. Lasseigne. 2005. Effects of KIBA on the rooting of stem cuttings of 15 taxa of snowbells ( Styrax spp.). J. of Envir on. Hort. 23:171-174. Grossmann, K. 2000. Mode of ac tion of auxin herbicides: a new ending to a long, drawn out story. Trends in Plant Sci. 5:506-508. 89

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Guo, X., Fu, X., Zang, D., and Y.M. 2009. Effect of auxin tr eatments, cuttings collection date and initial characteristics on Paeonia Yang Fei Chu Yu cutting propagation. Scientia Hort. 119:177-181. Haehle, R.G. and J. Brookwell. 1999. Nati ve Florida Plants: low maintenance landscaping and gardening. Gulf Publishing Company, Houston, T.X. Hammond, H.E., J.G. Norcini, S.B. Wilson, R.K. Schoellhorn, D.L. Miller. 2007. Growth, flowering, and survival of firewheel ( Gaillardia pulchella Foug.) based on seed source and growing location. Native Plants J. 8:23-37. Harper-Lore, B. and M. Wils on. 2000. Roadside Use of Nati ve Plants. Island Press, Washington, D.C. Harrington, J.F. 1972. Seed storage and longevity. In: T.T. Koslowski (ed.). Seed Biology vol. 3. Academic Press, New York, N.Y. Heather, A. E., Perez, H. P ., Wilson, S. B., Thetford, M., and Miller, D. L. 2009. Alleviating Seed Dormancy of Two Native Wildflowers: Polygonella polygama and Polygonella robusta. Southern Nursery Assoc. Res. Conf. Proc. 54:435-441. Hodges, A.W. and J.J. Haydu. 1999. Economi c Impact of Floridas Environmental Horticulture Industry, 1997. 30 August 2009. . Hodges, A.W. and J.J. Haydu. 2006. Economic impacts of the Flor ida environmental horticulture industry in 2005. 25 May 2009. < http://edis.ifas.ufl.edu/FE675>. Huegel, C.N. 1995. Florida Plants for Wildlife. Florida Native Plant Society, Spring Hill, F.L. Ingram, D.L. and T.H. Yeager. 1990. Propagat ion of landscape plants. 10 June 2009. . Jayasuriya, K.M.G.G., Baskin, J.M., Geneve, R.L. and C. C. Baskin. 2007. Morphology and anatomy of physical dormancy in Ipomoea lacunose : identification of the water gap in seeds of Convolvulaceae (Solana les). Ann. of Bot. 101: 341-352. Joshi, J., Schmid, B., Cald eira, M.C., Dimitrakopoulos, P .G., Good, J., Harris, R., Hector, A., Huss-Danell, K ., Jumpponen, A., Minns A., Mulder, C.P.H, Pereira, J.S., Prinz, A., Scherer-Lorenzen, M., Siamantziouras, A.-S. D., Terry, A.C., Troumbis, A.Y., and J.H. Lawton. 2001. Local adaptation enhances performance of common plant species. Ecol. Lett. 4:536-544. Justice, O.L. and L.N. Bass. 1978. Principl es and Practices of Seed Storage. Agr. Handbook No. 506. U.S. Government Printing Office, Washington, D.C. 90

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Kabat, S.M., Norcini, J.G. and B. Dehgan. 2007. Temper ature and light affects germination ecology of commercially produced seeds of Leavenworths coreopsis. Native Plants J. 8:236-247. Li, X., Baskin J.M., and C.C. Baskin. 1999. Physiological dormancy and germination requirements of several North American Rhus species (Anacardiaceae). Seed Sci. Res. 9:237-245. Lippit, L., Fidelibus, M.W., and D.A. Bai nbridge. 1994. Native seed collection, processing, and storage for revegetation pr ojects in the Western United States. Restor. Ecol. 2:120-131. Little, T.M. and F.J. Hills. 1978. Agricultural experiment ation: design and analysis John Wiley and Sons, New York, N.Y. Macdonald, B. 1986. Practical Woody Plant Propagation for Nursery Growers. Timber Press, Portland, O.R. Mack, R.N., and M. Erneberg. 2002. The United States naturalized flora: largely the product of deliberate introduc tions. Ann. of the Miss ouri Bot. Gardens 89:176. Marinelli, J., Ed. 1994. Goi ng Native: Biodiversity in our Own Backyards. Brooklyn Botanic Garden, Inc., Brooklyn. Miles, B. 1976. Wildflower Perennials fo r Your Garden. Hawthorn Books, Inc., New York, N.Y. Milstein, G. P. 2005. The uses and potential of wildflower seed in landscape. (eds.) M.B. McDonald and F.Y. Kwong. Flower Seeds: Bi olgoy and Technol.. CABI Publishing, Columbus, O.H. Metzger, J.D. 1992. Physiologica l basis of achene dormancy in Polygonum convolvulus (Polygonaceae). Amer. J. of Bot. 79:882-886. Meyer, S.E. and A. Paulsen. 2000. Chill ing requirements for seed germination of 10 Utah species of perennial wild buckwheat ( Eriogonum Michx. [Polygonaceae]). Native Plants J. 1:18-24. Myers, R.L. and J.J. Ewel, ( eds.) 1990. Ecosystems of Flori da. Univ. Presses of Fla. Gainesville. Nicolas, C., Nicolas, G., and D. Rodriguez. 1996. Antagonistic effects of abscisic acid and gibberellic acid on the breaking of dormancy of Fagus sylvatica seeds. Physiol. Planta 96:244-250. Norcini, J.G. and J.H. Aldrich. 2000. Cu tting propagation and contai ner production of Flora Sun' beach sunflower. J. of Environ. Hort. 18:185-187. 91

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Norcini, J.G. and J.H. Aldrich. 2004. Establis hment of native wildfl ower plantings by seed. 10 May 2009. < http:// edis.ifas.ufl.edu/EP227>. Norcini, J.G. and J.H. Aldrich. 2007. Stor age effects on dormancy and germination of native tickseed species. HortTechnol. 17:505-512. Norcini, J.G., J.H. Aldrich, and F.G. Martin 2001. Seed source ef fects on growth and flowering of Coreopsis lanceolata and Salvia lyrata J. of Environ. Hort. 19:212215. Norcini, J.G., Aldrich, J.H., and F.G. Mart in. 2006. Harvest season and fertilizer effects on seed production of Leavenworth's coreopsis. J. of Environ. Hort. 24:63-67. Norcini, J.G., J.H. Aldrich, L.A. Halsey and J.G. Lilly. 1998. Seed source affects performance of six wildflower species. Proc of Fla. State Hor t. Soc. 111:4-9. ORourke, F.L. 1940. The influence of blo ssom buds on rooting of hardwood cuttings of blueberry. J. of the Amer. Soc. for Hort. Sci. 40:332-334. Orozco-Segovia, A., Marquez-Guzman, J. Sanchez-Coronado, M.E., Gamboa De Buen, A., Baskin, J.M., and C.C. Baskin. 2007. Seed a natomy and water uptake in relation to seed dormancy in Opuntia tomentosa (Cactaceae, Opuntioideae). Ann. of Bot. 99:581-592. Osorio, R. 2001. A gardeners gu ide to Floridas native plant s. Univ. of Fla. Press, Gainesville. Paulson, A. Ed. 1989. Wildflower Resear ch Centers Wildflower Handbook. Texas Monthly Press, Austin. Perez, H.E., Almira, F., and M. Brennan. 200 9. Germination timing and dormancy break in seeds of summer farewell ( Dalea pinnata, Fabaceae). Ecol. Restor.27:160-168. Peters, J. (Ed.) 2000. Tetrazolium Testi ng Handbook. Associati on of Official Seed Analysts. Las Cruces, N.M. Pfaff, S., Gonter, M. A., Muara, C. 2002. Florida Native Seed Production Manual. 10 June 2009 . Phartyal, S.S., Kondo, T., Hoshino, Y. Baskin, C.C., and J.M. Baskin. 2009. Morphological dormancy in seeds of the autum-germinating shrub Lonicera caerulea var. emphyllocalyx (Caprifoliaceae). Plant Spp. Biol. 24:20-26. Phillips, H.R. 1985. Growing and Propagating Wildflowers. Un iv. of North Car. Press, Chapel Hill. 92

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PLANTS Database. Polygonella polygama PLANTS Profile. USDA 12 May 2008 . PLANTS Database. Polygonella robusta PLANTS Profile. USDA 12 May 2008 . Priestly, D.A. 1986. Implications for Seed Storage and Persistence in the Soil. Comstock Pub. Assoc., Ithica, N.Y. Puri, S. and R.C. Verma. 1996. Vegetative propagation of Dalbergia sissoo Roxb. using softwood and hardwood stem cuttings. J. of Arid Envi ron. 34:235-245. Rosier, C.L., Frampton, J., Goldfarb, B., Blazich, F.A., and F.C. Wise. 2004. Growth stage, auxin type, and concentration influenc e rooting of stem cuttings of Fraser fir. HortScience 39:1397-1402. Rukuni, D. 2008. Dormancy in pr e-variety germplasm of native Coreopsis species. PhD Dissertation. Dept. of Environ. Hor t., Univ. of Fla ., Gainesville. Scheiber, S.M., Gilman, E.F. Sandrock, D.R., Paz, M., Wiese, C. and M.M. Brennan. 2008. Post-establishment landscape perfo rmance of Florida native and exotic shrubs under irrigated and non-irrigated conditions. HortTechnol. 18:59-67. Sharma, J. and W.A. Grav es. 2005. Propagation of Rhamnus alnifolia and Rhamnus lanceolata by seeds and cuttings. J. of Environ. Hort. 23:86-90. Sharma, S. D. and N. B. Aier 1989. Seasonal rooting behav iour of cuttings of plum cultivars as influenced by IBA treatm ents. Scientia Hort. 40: 297-303. Taiz, L. and Zeiger, E. 2006. Plant Physiology, 4th ed. Sinauer Associates, Inc., Sunderland. M.A. Tallamy, D.W. 2004. Do alien plants reduc e insect biomass? Conserv. Bio. 18:16891692. Tang, A.J., Tian, M.H., and C. L. Long. 2009. Environmental control of seed dormancy and germination in the short-lived Olimarabidopsis pumila (Brassicaceae). J. of Arid Environ. 73:385-388. Taylor, W.K. 1998. Florida wildflowers in thei r natural communities. Univ. Press of Fla., Gainesville. Thetford, M., Heather, A.E., Perez, H.E., and S.B. Wilson. 2008. Propagation of native wildflowers from wild collected seeds or cuttings. Inter. Plant Prop. Soc. Proc. 58:55-560. Thetford, M., Miller, D., and P. Penniman. 2001. Vegetative propagation and production of Ceratiola ericoides Michx. for use in restorati on. Native Plants J. 2:116-125. 93

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94 Turner, S.R., Merritt, D.J ., Baskin, C.C., Dixon, K.W. and J.M. Baskin. 2005. Physical dormancy in seeds of si x genera of Australian Rhamnaceae. Seed Sci. Res. 15:51-58. U.S. Fish and Wildlife Service. 2001. U.S. Fish and Wildlife Service Trasmittal Sheet. 22 June 2009 < http://www.fws. gov/policy/601fw3.pdf> Valero-Aracama, C., Wilson, S.B., Kane, M. E., and Philman, N.L. 2007. Influence of in vitro growth conditions on in vitro and ex vitro photosynthetic rates of easyand difficult-to-acclimatize sea oats ( Uniola paniculata L.) genotypes. In Vitro Cell. and Develop. Bio. 43:237-246. Vandelook, F., Bolle, N., and J.A. Van A ssche. 2009. Morphological and physiological dormancy in seeds of Aegopodium podagraria (Apiaceae) broken successively during cold stratification. Seed Sci. Res. 19:115-123. Vora, R.S. 1989. Seed germinati on characteristics of selected native plants of the lower Rio Grande Valley, Texas. J. of Range Manage. 42:36-40. Walton, D. and L. Schiller. 2007. Natural Florida Landscapi ng. Pineapple Press, Inc., Sarasota, F.L. Widrlechner, M. 2007. While they were asleep: do seeds after-ripen in cold storage? Experiences with Calendula. Intl. Plant Prop. Soc. Proc. 57: 377-382. Wildflower Seed and Plant Growers Associat ion, Inc. 2003. About Us. 30 September 2009 < http://www.floridawildflowers.com/about.htm> Willis, A.J. and R.H. Groves. 1991. Temperature and light effe cts on the germination of 7 native forbs. Aust. J. of Bot. 39:219-228. Wunderlin, R.P. and B.F. Hansen. 2003. Guide to the Vascular Plants of Florida, 2nd ed. Univ. Press of Fla., Gainesville.

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BIOGRAPHICAL SKETCH Alison graduated from the University of Florida in 2007 with a Bachelor of Science degree in Environmental Horticulture. She obtained her Master of Science degree in December of 2009 in the same department with a focus on Plant Restoration and Conservation. 95