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16. Unlocking Milkweeds Secret Code - Hybridization
08/31/2025
16. Unlocking Milkweeds Secret Code - Hybridization
This compilation of excerpts details Mary Lewis's doctoral dissertation from the University of Georgia (2021), focusing on the hybridization of Asclepias species to create new cultivars. The research addresses the commercial limitations of existing milkweed varieties, such as minimal branching and height issues, by optimizing pollination methods, including a novel inverted pollinia technique. Lewis explores interspecific hybridization using Asclepias tuberosa as the maternal parent, successfully developing hybrid seeds with several other Asclepias species and analyzing inheritance patterns for traits like flower color, pubescence, sap color, foliage, and height. Additionally, the dissertation establishes effective vegetative propagation protocols for A. tuberosa and develops an embryo rescue protocol to overcome seed abortion issues, along with assessing genomic content across various Asclepiasspecies to understand hybridization barriers. The ultimate goal is to improve the commercial viability and conservation efforts for this vital pollinator genus. Pre-18th Century: Asclepias (Milkweed) Species Diversity and Distribution: Asclepias genus, a vital pollinator genus and host plant for monarch butterflies, comprises over 100 species in North America, with 90 species throughout the U.S., Canada, and parts of Central America. This broad native range leads to significant phenotypic diversity and often species isolation. Traditional Milkweed Propagation: In the wild, Asclepias species reproduce via seeds and rhizomatous tubers. Understanding Asclepias Floral Structure: The genus is known for one of the most complex reproductive structures in the plant kingdom, similar to orchids, transmitting pollen via sac-like pollinia in a "lock and key" method. This complexity encourages outcrossing and limits natural interspecific hybridization due to varying pollinia and stigmatic slit sizes. Self-Incompatibility: Asclepias species carry an S-locus gene promoting self-incompatibility, which is late-acting in the ovary and hinders endosperm maturation, often leading to seed pod abortion if less than half the embryos are fertilized. Limited Commercial Production: Most Asclepias species lack commercially desirable traits like good branching, suitable height, and wide color variation, limiting their presence in the ornamental market. Late 18th Century: First Documented Embryo Rescue: Charles Bonnet performs the first recorded embryo rescue, setting groundwork for future plant breeding techniques. Early 20th Century (1900s-1940s): 1904: Hannig refines embryo rescue techniques. 1922: Knudson demonstrates drastically improved germination rates for orchid seeds by prematurely harvesting pods for embryo rescue. 1923: C.B. Hutchings notes the monarch butterfly's migratory habits and dependence on milkweed. 1940: E.D. Putt's observations on sunflower morphology, including branching. 1945: O.A. Stevens documents successful controlled hybridization between A. speciosa and A. syriaca. 1948: Heiser and Whitaker document chromosome numbers in California weeds, including some Asclepias species. 1948: Sparrow and Pearson study pollen compatibility in Asclepias syriaca, noting issues with endosperm fertilization failure leading to embryo starvation. Mid-20th Century (1950s-1970s): 1950: Swift defines C-value (total genomic content). 1954: R.E. Woodson publishes "The North American Species of Asclepias L.", noting 106 species. 1956: Darlington and Wylie's "Chromosome Atlas of Flowering Plants" lists several Asclepias species as diploids. 1960: R. Allard publishes "Principles of Plant Breeding," discussing inheritance of quantitative traits. 1969: J.R. Nolan studies bifurcation of the stem apex in Asclepias syriaca, showing its limited branching capacity. 1970: G.L. Stebbins describes the "lock and key" pollination method in Asclepias. 1972: Evetts and Burnside classify A. tuberosa seedling dormancy as primary, broken by cold-moist stratification, seed coat removal, or Gibberellic Acid. 1976: R. Wyatt publishes "Pollination and fruit-set in Asclepias: a reappraisal," and H.M. Wilbur studies life history evolution in seven milkweed species. 1977: Baskin and Baskin note A. syriaca requires extended cold stratification (a year or more) for good germination. Late 20th Century (1980s-1990s): 1980: Kephart and Heiser report successful controlled hybridizations in Asclepias. 1981: S.R. Kephart studies breeding systems in Asclepias incarnata, A. syriaca, and A. verticillata. R. Wyatt also studies the reproductive biology of A. tuberosa. 1984: Bookman provides evidence for selective fruit production in Asclepias, noting pod abortion if less than half the seeds are fertilized. 1986: T.R. Shannon and R. Wyatt detail the juvenile period and flower production in A. exaltata. 1988: Kephart, Wyatt, and Parrella publish on hybridization in North American Asclepias, including successful crosses like A. exaltata x A. quadrifolia and A. purpurascens x A. syriaca. 1991: R. Wyatt and D.M. Hunt report on hybridization in North American Asclepias using flavonoid evidence, including A. exaltata x A. syriaca. Malcolm studies cardenolide-mediated interactions between plants and herbivores. Albrecht and Lehmann show daylength, cold storage, and production method influence A. tuberosa growth and flowering. 1992: R. Wyatt and S.B. Broyles publish on hybridization in North American Asclepias using isozyme evidence, suggesting habitat overlap influences natural hybridization. 1993: Ecker and Barzilay discuss propagation of Asclepias tuberosa from short root segments, noting optimal conditions for rooting. 1994: M.P. Bridgen reviews plant embryo culture. R. Wyatt and S.B. Broyles review the ecology and evolution of reproduction in milkweeds. 1999: Zalucki and Malcolm study plant latex and monarch larval growth/survival. Early 21st Century (2000s-2010s): 2000: S.R. Lipow and R. Wyatt identify single gene control of postzygotic self-incompatibility in A. exaltata. W. Cullina publishes "Wildflowers: A Guide to Growing and Propagating Native Flowers of North America." 2002: S.B. Broyles studies hybrid bridges to gene flow in milkweeds, specifically A. exaltata and A. syriaca. 2004: Klips & Culley confirm natural hybridization between A. syriaca and A. sullivantii at an Ohio site. 2007: Doležel, Greilhuber, and Suda publish on estimating nuclear DNA content using flow cytometry. 2009: Agrawal and Konno provide a model for understanding latex in plant defense. Agrawal et al. study phylogenetic ecology of leaf surface traits in milkweeds, including pubescence. 2011: Fishbein et al. infer phylogenetic relationships of Asclepias from chloroplast DNA sequences. 2012: Bai et al. report new nuclear DNA content for vascular plant taxa, including some Asclepias species. J.M. Pleasants and K. Oberhauser study milkweed loss and its effect on monarch populations. 2013: Luna and Dumroese review monarch and milkweed interactions and propagation methods. Shearer and Ranney study ploidy levels and genome sizes in dogwood. 2015: K. Weitemier et al. study intragenomic polymorphisms in Asclepias nuclear ribosomal DNA. 2018: Kaye et al. study seed dormancy and germination in milkweeds. 2018: October: Mary Lewis's research team initiates germination of various Asclepias species for parent production at the University of Georgia (UGA) Athens campus. March - October: Mary Lewis's team conducts approximately 7,000 interspecific pollination attempts using an "inverted pollinia method" (optimized by Lewis's team) between A. tuberosa (maternal parent) and seven other Asclepias species. This results in 253 hybrid pods. May - November: Initial germination and growth of hybrid seedlings. June 18: Vegetative cuttings are harvested from mature A. tuberosa stock plants (six weeks after dormancy break) and juvenile seedlings (14 weeks after germination) for propagation experiments. July: Mary Lewis's team develops an Embryo Rescue (ER) protocol for A. tuberosa, including optimal media type (½ MS) and embryo maturity (60 days after pollination, DAP). This is the first documented ER protocol for Asclepias tuberosa. November - February 2019: Hybrid seed germination (both direct-seeded and stratified) continues. 2019: July: A significant spider mite and thrip infestation impacts the hybrid population, requiring some plants to be cut back and placed in cold storage. Throughout the year: Mary Lewis's team collects initial data on hybrid traits (juvenile stage). 2020: January: Mary Lewis's team initiates germination of 15 Asclepias species for genome size assessment. April: Nathaniel P. Hofford submits his undergraduate research thesis on admixture between A. syriaca and A. sullivantii to Ohio State University, finding distinct morphological features and possible extensive admixture. July: Mary Lewis's team completes data collection on remaining hybrid plants (mature stage) after they are removed from cold storage and re-grown. Throughout the year: Flow cytometry is used to assess the total genomic content of 15 Asclepias species and four hybrids, documenting a 2C genome size range of 0.65 to 1.24 picograms. 2021: February: Mary Lewis submits research on the inheritance of qualitative traits (pubescence, sap color, flower color) and quantitative traits (height, leaf length, leaf width, flower height, branching) in Asclepias interspecific hybrids for review. July 29: UGA research on milkweed breeding, led by Mary Lewis and Matthew Chappell, is featured on the cover of HortScience. The article highlights the first successful intentional interspecific hybridization of milkweed species and the potential for novel cultivars. Ongoing: Mary Lewis's research aims to continue to improve commercial production and conservation efforts for Asclepias species. Research by Hofford and his advisors seeks to further clarify the degree of genetic exchange between A. syriaca and A. sullivantii and its conservation implications. Apogee Instruments Case Study highlights the importance of ecological impact assessment for new native plant cultivars and details the use of ePAR sensors in Asclepias hybrid research. Cast of Characters Researchers & Academics: Mary Lewis: A doctoral candidate at the University of Georgia (UGA) under the direction of John Ruter. She led the pioneering research on interspecific hybridization, vegetative propagation, embryo rescue, genome size assessment, and trait inheritance in Asclepias species, culminating in a detailed dissertation and several published articles, including a cover feature in HortScience. She received her BS from the University of Georgia in 2017 and her PhD in 2021. John Ruter: Mary Lewis's Major Professor at the University of Georgia. He stepped in as a committee member and major advisor after the passing of Dr. Paul Thomas and the departure of Lewis's previous advisor, guiding her to the completion of her Ph.D. Matthew Chappell: Horticulture Professor at the University of Georgia and a key researcher on Mary Lewis's team. He is also the corresponding author for some of the published research. Paul Thomas: The late Dr. Paul Thomas was instrumental in convincing Mary Lewis to pursue graduate school and was her initial biggest supporter and committee member. His motto, "we do not fail," inspired her work. Donglin Zhang: A committee member for Mary Lewis's dissertation. He contributed critical resources and lab space for culture and excising in the embryo rescue project. Ali Missaoui: A committee member for Mary Lewis's dissertation. Ockert Greyvenstein: A committee member for Mary Lewis's dissertation and Senior Plant Breeder at Ball Horticultural Company. He tutored Mary Lewis, exposing her to and fostering her love for plant breeding. Ron Walcott: Vice Provost for Graduate Education and Dean of the Graduate School at the University of Georgia in December 2021, electronic version approved Mary Lewis's dissertation. Nathaniel P. Hofford: An undergraduate researcher at The Ohio State University. He conducted a research thesis on the admixture between Asclepias syriaca and Asclepias sullivantii, using morphological and genomic data. Dr. Stephen M. Hovick: Project Advisor for Nathaniel P. Hofford's undergraduate research thesis at The Ohio State University. He collected Iowa samples and provided continuous advice and guidance. Dr. Andreas S. Chavez: Project Advisor for Nathaniel P. Hofford's undergraduate research thesis at The Ohio State University. He provided invaluable advice regarding genomic analyses and ddRADSeq execution. Dr. Robert "Bob" Klips: His previous study (Klips & Culley, 2004) inspired Nathaniel Hofford's research. He also contributed to Hofford's study by collecting individuals and sharing information about Asclepias hybridization. Dr. Kattia Palacio-Lopez: Offered advice and helped with ddRADSeq preparation for Hofford's study. Colin Day: Collected individuals, extracted hundreds of Asclepias DNA samples, and helped with ddRADSeq preparation for Hofford's study. Kali Mattingly: Sampled from Ohio, Wisconsin, and Illinois populations and provided constant advice throughout Hofford's project. Aaron Tyal: Assisted in the collection of individuals from several Ohio populations for Hofford's study. Jimmy Bingman, Mounica Burada, Allie Falla, Austin Kramer, Bailey Madercic, Selinde Roosenburg, and Lizzy Slabinski: Helped with morphological data collection and DNA quantification for Hofford's study. Justin Peterman, Conor Fair, Hannah Brown, Kris Braman, Marc W. van Iersel: Authors of the Apogee Instruments case study on the ecological impacts of milkweed hybrids, all associated with UGA's College Station Greenhouse. Pam Lewis: Assisted Mary Lewis in keeping her plants alive over four years, providing crucial support with plant care, pest control, and greenhouse maintenance. Rebekah Maynard: A student worker and graduate research assistant who was instrumental in Mary Lewis's success, assisting with data collection and plant potting. She is also a co-author on several publications. Justin Peterman: A student worker who was instrumental in Mary Lewis's success, assisting with data collection and plant potting. Carver Nichols: A student worker who was instrumental in Mary Lewis's success, assisting with data collection and plant potting. William English: A student worker who was instrumental in Mary Lewis's success, assisting with data collection and plant potting. Alana Edwards: A student worker who was instrumental in Mary Lewis's success, assisting with data collection and plant potting. Ryan McNeil: Assisted Mary Lewis in developing an embryo rescue and tissue culture methodology within the Merkle lab. Dr. Merkle: Assisted Mary Lewis in developing an embryo rescue and tissue culture methodology within the Merkle lab. Dr. Heather: Assisted Mary Lewis in developing an embryo rescue and tissue culture methodology within the Merkle lab. Rachael White: A friend and colleague of Mary Lewis at UGA, providing friendship, input, expertise, and study partnership. Sean Caton: A friend and colleague of Mary Lewis at UGA, providing friendship, input, expertise, and study partnership. Dr. Lombardini: A professor in the Horticulture Department at UGA who taught Mary Lewis skills for her future. Dr. van Iersel lab: The lab of Dr. Marc W. van Iersel, which contributed to Mary Lewis's education and research. Organizations & Companies: University of Georgia (UGA): The primary institution where Mary Lewis conducted her Ph.D. research and where much of the milkweed breeding, propagation, and ecological impact studies took place. Ball Horticultural Company: Ockert Greyvenstein, a senior plant breeder, is associated with this company and contributed to Mary Lewis's research. The Ohio State University: Institution where Nathaniel P. Hofford conducted his undergraduate research on Asclepias hybridization. Apogee Instruments: Manufacturer of ePAR sensors used in ecological impact studies of milkweed hybrids at UGA, and featured in a case study about the research. I. Quiz: Short Answer Questions Answer each question in 2-3 sentences. What is the primary ecological importance of Asclepias species? Asclepias species are vital pollinator plants across North America and serve as the exclusive host plant for monarch butterfly larvae. They provide nectar for adult butterflies and serve as oviposition sites for eggs, with caterpillars feeding solely on Asclepias foliage. Why is commercial production of Asclepias limited, despite its ecological value? Commercial production is limited because most Asclepias species lack commercially desirable traits. These undesirable traits include minimal branching habits, excessive height, and limited color variation, making them difficult to produce, rack, and ship efficiently. Describe the novel "inverted pollinia method" and its advantage in Asclepias hybridization. The inverted pollinia method involves inserting the pollinia perpendicular to the stigmatic slit, resembling a key in a lock. This technique places the pollinia in direct contact with the stigmatic surface, increasing pollination success rates fourfold compared to traditional methods for intraspecific crosses. Which Asclepias species successfully formed hybrid seeds with A. tuberosa as the maternal parent in the Lewis study? Four species successfully developed hybrid seeds with A. tuberosa: Asclepias hirtella, Asclepias purpurascens, Asclepias speciosa, and Asclepias syriaca. These species were generally found to be more genetically similar to A. tuberosa. What was the most effective germination technique for hybrid Asclepias seeds, and why is this understandable given their native ranges? Cold-moist stratification for 30 days had the highest germination rates across the four hybrid groups. This is understandable because all successfully hybridized species have native ranges in northern climates with distinct dormant winter seasons, necessitating a cold treatment to break seed dormancy. What is "embryo rescue" and why is it a valuable technique for Asclepias breeding? Embryo rescue is the isolation and in vitro growth of immature embryos under sterile conditions to obtain viable plants. For Asclepias, it's valuable because it can bypass late-term embryo abortion, a common reason for hybridization failure, by providing essential nutrients that might otherwise be lacking due to endosperm fertilization failure. How does the timing of embryo rescue impact its success and efficiency for Asclepias tuberosa? For A. tuberosa, rescuing embryos at 60 days after pollination (DAP) proved optimal for root and shoot growth without needing plant growth regulators. This significantly reduces propagation time by 88 days compared to traditional methods, as embryos are autotrophic at this stage. What is the C-value, and how does it relate to Asclepias hybridization success? The C-value characterizes total genomic content, measured in picograms of DNA in a whole chromosome complement. In Asclepias, species with significant differences in genome size generally experienced difficulty forming hybrids, suggesting that genome size can be an indicator of potential hybridization success. Describe the inheritance pattern of sap color in the Asclepias hybrids studied. White sap color was found to be a dominantly inherited trait across all hybrid groups, consistently expressed at a ubiquitous 1:0 ratio (white sap to clear sap). Clear sap, characteristic of the maternal parent A. tuberosa, was recessively inherited. What type of inheritance pattern was generally observed for quantitative traits like height and leaf length in the F1 Asclepias hybrids? For quantitative traits such as height, leaf length, and leaf width, inheritance generally resulted in values intermediate of the parents. While not always fitting a statistically normal distribution, the progeny typically exhibited a wide range of values...
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