Daylily: Botany, Propagation, Breeding

oft

Surinder K. Gulia, Bharat P. Singh, and Johnn y Carter Agriculture Research Station Fort Valley State University Fort Valley, GA 31030 USA

Robert I. Giiesbach United States Department of Agriculture Agricultural Research Service Beltsville, MD 20705 USA

I. INTRODUCTION II. BOTANY A. History B. Systematics III. ANATOMY AND PHYSIOLOGY A. Roots. Stains, and Leaves B. Inflorescences and Flowers IV. HORTICULTURE A. Propagation B. Pests and Diseases C. Coltivar Registration and Awards V. GENETICS A. Genorne and Ploid y Level B. Flower Color Inheritance C. Biotechnology VI. CONCLUSION VII. LITERATURE CITED

I. INTRODUCTION

Davlilies (Hernerocallis spp., Hemerocallidaceae) are herbaceous perennials grown extensively as ornamental plants in home gardens

Horticultural Reviews. Volume 35 Edited by Jules Janick Copyright L 2009 John Wiley & Sons, Inc.

193 194 S. K. GULIA, B. P. S]NGH. J. CARTER. AND R. J. GRIESBACH

and commercial land inflorescences worldwide. Their botanical name, Hernerocallis, is derived from the Greek words hernero ("a day") and callis ("beauty") referring to the fact that each flower lasts only one day (Panavas et al. 1999). However, multiple buds on the inflorescences provide bloom over a number of weeks. Ancient Chinese and Japanese used daylilios for their roots, leaves, and flowers as both food and medicine. Daylily buds contain more protein and vitamin C than green beans and asparagus and vitamin A equivalent to asparagus (Erhardt 1992). All parts of the plant are edible and consumed either dried or fresh (e.g., young shoots are cooked as vegetables in China while flowers and bud are delicacies in the cuisines of several southeast Asian countries). Daylily can be consumed in various preparations, such as chicken with davlily, daylily soup, daylily casserole, deep-fried daylilies, and steamed daylily. Recent sensory evaluation and consumer preference studies (Knight et al. 2004; Pollard et al. 2004) also support the potential food value of davlilies. In addition, daylily roots and crowns are used as a pain reliever, a diuretic, an antidote to arsenic poisoning, and an anticancer agent (American Hemerocallis Society 2007). Daylily flowers are known to possess antioxidant properties (Mao et al. 2005) and cyclooxygenase inhibitory activities (Cichewicz and Nair 2002). Daylilies are easy to grow, have attractive flower colors and shapes, and the plant has an attractive growth habit. They are tolerant to drought, flooding, and heat stress and grow well in most soil types under full sun or light shade. Besides of their esthetic value, they are used to help control soil erosion along highways and water channels (Munson 1989; Garber 2004). Daylilies thrive well over a large climatic range in North America, from southern Florida to northern Canada (Peat and Petit 2004), Daylilies, however, are unsuitable as cut flower or potted plant due to short flower life. Market value of daylilies in United States together with other perennials was estimated at $571 million in 2002 (U.S. Dept. of Agriculture [USDA] 2003).

II. BOTANY

A. History

According to Chinese oral traditions, reference to da ylily (known as Hsuan Ts'ao) dates back to 2697 BCE (Kitchingman 1985), but the first written record appears in the canonical writings of Confucius dating 3. DAYLILY: BOTANY, PROPAGATION, BREEDING 195 back to about 551-479 BCE (Barnes 2004). Hu (1968a) found the first reference of H. fulva in the writing of the Chou dynasty dating back to 112-255 BCE, where it was grown for food and medicine (Kitchingman 1983). About 300 BCE, daylily was brought from the Far East to Europe by the silk and spice traders. By 25 BCE, ii. flava was known to the Greeks, Romans, Egyptians, and Africans (Baker 1937). In 1597, John Gerard was the first English herbalist who used the name daylily to designate the Chinese Hsuan Ts'ao (Hu 1968a). By 1620, H. flava and H. fulva daylilies were cultivated in England (Stout 1934), they found their way to the United States in the 18th and 19th centuries (Garber 2004), The pre-Linnnaean name of da ylil y was Ephemeron from the Greek epi ("upon') and hemera ("day"). In 1753, Linnaeus retained hem era and added cabs ("beauty") and created a new generic name Hemerocalbis (Eddison 1987). Many books describe the early history of daylily including information on species classification, propagation, and cultural practices (Stout 1986: Munson 1989; Erhardt 1992). Schabell (1990) has reviewed the history of daylily cultivation in detail.

B. Systematics Hemerocallis is native to Asia throughout China, northern India, Japan, and Korea. The first extensive taxonomical study of Henleroca ills was carried out by Stout (1941); however, he died before his complete monograph could be published. Using Stout's draft manuscript, Shiu- Ying Hu (1968b) published the first monograph with a key to 23 species separated into three groups. In 1969, Hu recognized two additional species: H. tazaifu (Hu 1969a) and H. darrowiana (Hu 1969b). Erhardt (1992) developed a more elaborate classification of daylily species separating them into five groups: fulva, citrina, middendorfli, nana, and multiflora (Table 3.1). Erhardt recognized only 20 species (Table 3.2). He did not recognize H. gramiflea, H. luteola, or H. littorea. Since 1992, two additional species have been recognized: I-I. hongdoensis (Chung and Kang 1994) and H. taeanensis (Kang and Chung 1997). In 1985, Dahlgren et al. separated Heinerocallis from the Liliaceae and placed them within their own family, the Hemerocallidaceae. Homer- ocallidace'ae differ from Liliaceae in the shape of their seeds, placement of their nectarines, and type of their roots. Hemerocallidaceae seeds are black and round shaped while Liliaceae seeds are brown and flat. Nectaries are located in the walls of the ovary in Hernerocallidaceae but

196 S. K. GULIA. B. P. SINGH. J. CARTER. AND R. J. GRIESBACH

Table 3.1. Classification of Hemerocollis species and common characteristics of each group. Species group Species Common characteristics

Citrina H. oltissima. H. citrina, Inflorescences are multiple branched, H. coreano, H. lilioasph ode/us. flowers mostly yellow, nocturnal H. minor, H. pedicellata. habit, fragrant with long perianth H. thunbergii, H. yezoensis tubes.

Fulva H. x aurantiaca, ii. fulva Inflorescences are branched, flowers brownish-red (fulvous d ye) color, diurnal habit, roots have spindle- shaped swellings. Middendorffii H. dumoi'tieri. H. esculcnto, Inflorescences are noobranched, i-I. exoltata, I-I. hakuunensis, flowers orange color, diurnal habit. II. middendorffii bracts short, broad and overlapping.

Nana H. forrestii, H. nano Inflorescences are nonbranched, inflorescences max. 50 cm long, flowers reddish-orange color, diurnal habit, perianth tube shorter than I cm; not winter-hardy.

Multiflora H. micron thu. H. in ultiflom. Inflorescences have man y branches, H. plicata orange to orange-yellow color, diurnal habit, flowers on short stalks smaller than 7 cm. tubes less than 2 cm long. Source: Erhardt 1992. at the base of the perigonial leaves in Liliaceae, Unlike Liliaceae, Hemerocallidaceae do not grow from true bulbs. Molecular approaches are helping to more accurately define Hemerocallis taxa (Noguchi and De-Yuan 2004; Noguchi et al. 2004). For example, it appears that H. citrina var. vespertina originated from at least three different lineages that invaded the Japanese archipelagoes separately through different routes (Noguchi and De-yuan 2004). Juerg Plodeck and Jianping Zhuang Plodeck (2003) have a Web site that describes Hemerocallis systematics in detail (www.hemerocallis-species .coin/).

III. ANATOMY AND PHYSIOLOGY

The anatomy and physiology of daylily plants was reviewed in detail by Voth et al. (1968). Daylily plants are composed of rammets, commonly called fans, that consist of an underground thickened stern, roots, rhizomes, leaves, and flowering inflorescences. The underground stern, commonly called a crown, contains the apical meristem. The crown is 3. DAYLILY: BOTANY. PROPAGATION. BREEDING 197

Table 3.2. List of Hemerocallis species with an overview of their traits description. Name of species Description of traits H. aitissima M—La: nor.: nbc., fr.; flowers on inflorescence 120-200cm tall: Pale yellow flower; >30 flowers per inflorescence; flower diani. 7.5 cm: tall; fading in hot son I-I. a wan tiaca EM: cv.: din.; flowers on inflorescence 60-90 cm tall; orange with red tinge flower; 6-8 flowers per inflorescence; flower diameter > 12 cm II. auvantioca Major' EM; cv.; dio.; sI. fr.; ext.: Re.; flowers on inflorescence 60-90 cm tall; yellow-orange; 5-10 flowers per inflorescence; flower diameter >12cm IL citrina M—MLa; dor.; nor..; flowers on inflorescence 100-115 cm tall; pale yellow flower; 30-70 flowers per inflorescence; flower diameter > 12cm I-I. citrina var. M—MLa; dor.; nbc., sl.fr .; flowers on inflorescence 180 cm tall: vespertina light yellow flower: 30-70 flowers per inflorescence: fading in full sun H. coreana EM—M; dor.: din.; fr.; flowers on inflorescence 50-80 cm tall: yellow flower: 50-80 flowers per inflorescence II. darrowiana M—MLa: dor.; diu.; yellow flower: 2 flowers per inflorescence H. duioortjeii EE ; dor.; dio.; flowers on inflorescence 15-60 cm II: it flower; sepal outside brownish red: 2-4 flowers i inflorescence I-I. esciileota EM; dor.; diii.; flowers on inflorescence 60-90 cm I:! flower; 5-6 flowers per inflorescence H. exaltata EM—M; dor.; din.: flowers on inflorescence 120-150 cin tall: orange flower: branching only at its apex; thick inflorescences, recorving sepals, and broad petals H. foricstii EM; dor.; dio.: flowers on inflorescence 30-40 cm tall; orange-red or orange (two forms): pedicel 2-3 cm long, not everywhere winter-hardy: low growing: pedicels i-I. fulva EM; dor.; din.: flowers on inflorescence 60-90 cm tall: orange with red tinge; H. fulva 'Kwanso' and II. folva 'Flare Pleno' have double flowers: Ir. fulva var. rosen has rose-red flowers; H. folva var. Iitforea shows semi-evergreen to evergreen behavior: redurving tepals; eye, bitone, wavy margins; median stripe; nerves: folvoos-red and rose: medium to long tube: a few IL fulva varieties can show op to 100 flowers per inflorescence IL gralninea EM; dor.: diu., ext.; flowers on inflorescence up to 75 cm tall: strong orange flower: 2-3 flowers per inflorescence; flower diameter > 10 cm; grasslike leaves H. hokoonensis M; dor.; dio.; flowers on inflorescence 85-100 cm tall: orange, flowers 6-11 flowers per inflorescence II. hangdoensis M: dor.; diii.; flowers on inflorescence 60-90 cm tall; orange-yellow flower; 1-cm-long pedicel; 5-17(-23) flowers per inflorescence

(continued) 198 S. K. GULIA, B. P. SINGH, J. CARTER, AND R. J. GRIESBACH

Table 3.2. (Continued) Name of species Description of traits

H. lilioaspliodclus E-EM; dor.; noc.—dio., fr., ext.; flowers on inflorescence 76 cm tall: light yellow flower; makes runners, trumpet form H. rniciantlia —; dor,: diu.; - cm; orange flower; 4 flowers per inflorescence; very small tepals H. iniddendorffii EE; dor.; diu.; Re.; flowers on inflorescence 60-90 cm tall; orange flower; the 2 bracts are broad oval and overlapping at base; dwarf plant; up to 10 flowers per inflorescence H. minor E—EE; dor.; diu.; fr.; ext.; flowers on inflorescence 45-60 cm tall; yellow flower; pedicels several cm long; dwarf habit; grasslike leaves; 2-5 flowers per inflorescence H. multi,flora M—MLa; dor.: din.; flowers on inflorescence 60-120 cm tall; orange flower; 75-100 flowers per inflorescence; repeatedly branched; 0.8-1 .2-cm-long pedicels: broad petals: trumpet shape 11. none E; dor.; diu.; flowers on inflorescence 15-30 cm tall; reddish- orange flower; only 1 flower per inflorescence; very small tube; not everywhere winter-hard y: dwarf pedicellata — ; dor.; diu.; flowers on inflorescence 55-65 cm tall: red-orange flower; pedicel length 2-4.5 cm H. p/irate E -; dor.; dio.; flowers on inflorescence 25-55 cm tall: orange-yellow flower; 0.5-2 cm long pedicels: 5-11 flowers per inflorescence if. fneanenj.c EM; dor.; din.; flowers on inflorescence 30-70 cm tall; orange-yellow flower; 0.2-3-cm-long pedicels Ii. llnzuhinj1 \l—MLa; dor.; nec.; fr.; ext.; flowers on inflorescence 100-115 ciii tall; lemon-yellow flower: green throat; flower diameter up to 10 cm; 1-2-cm-long pedicels: 4 .20 flowers per inflorescence; ruffled tepals and broad petals IL 11ZO('flSIS — : dor.; diu., sift.: flowers on inflorescence 40-85 cm tall: lemon-yellow flower; flower diameter up to 10 cm; op to 3-cm-long pedicels; 4-12 flowers per inflorescence I lower time: EE extra early; E = early: EM - early midseason; M - midseason; MLa - We midseason; La = late; VLa very late; Winter behavior: dor. = dormant, deciduous; sev. .--- semi-evergreen; iv. evergreen; I- lower characteristics: noc. nocturnal; diii. diurnal; si. fr. = slightly fragrant; ft. = trnirant; ext. = extended; dbl. - double; Re. rebluom. - Information unavailable. 111 101'. l-r/i(Ii-lJt 1902 Ploiliik 2002.

3. DAYLILY: BOTANY, PROPAGATION, BREEDING 199

Bloom scar,, Seed pod Bud

-;:\ J/9—\\c\ ., Pistil Stamen ,, " Spent bloom Midrib • Throat (p/i Petal Sep:

Proliferation ^)j/- Scape

Foliage

Stigma - = Style Anther Pistil cc CrownCro [Filament_ ( /Ovaryj Roots

Fig. 3.1. Davlilv plant showing various parts with emphasis on floral biology. (Sourco: Iowa State Universit y, 2006. Universit y Extension publication available at www. extension.iastate.edu/Publications/RG303.pdf#search--22RG303.)

top-shaped and develops a shallow depression on its upper surface (Voth et al. 1968). The leaves and roots arise from this depression. Contractile roots keep the crown underground (Putz 1998). A typical daylily plant is illustrated in Fig. 3.1.

A. Roots, Stems, and Leaves Daylily roots can be fibrous or rhizornatous (Voth et al. 1968). The difference in the root system is one criterion for separating daylilies into different species. The roots may form flesh y tuberlike structures, as in case of H. citrine. In H. minor and H. nana, the roots thicken only near their ends, indicating that these species are related to each other. 200 S. K. GLLIA. B. P. SINGH, J. CARTER. AND R. J. GRIESBACH

Roots of H. dumortieri are cylindrical; those of H. fulva are spindle shaped. Daylilies are drought-resistant because of two root characteristics: The rhizomatous roots can store large amount of water and fibrous roots can exploit soil water fully. Putz (1998) describes a contractile behavior of H. fulva roots pulling the cryptocorm down- ward, thus not allowing the roots to get too close to the soil surface arid be exposed to harsh weather conditions while dormant. Daylily leaves cannot be divided into distinct blade, petiole, and base regions (Voth et al. 1968). In general, they are grasslike in appearance and are arranged "fanlike" in two compact ranks (distichous). The leaves may stand erect, arch outward, or bend over near their tips or tend to fold along midrib. Daylilies exhibit three types of growth habit commonly referred to as dormant, semi- evergreen, and evergreen. Dormant types resume growth in spring when day temperature becomes warm enough to support growth and stop growing in autumn when days shorten and temperature cools. Before leaves die down, a compact resting bud is formed in the crowns of the plant. Within the compact bud, the leaves are protected from freezing and dehydration during winter. Dormant behavior in daylilies can be induced by short day-length or low temperature. In the fall, one or more axillary vegetative buds are formed, which remain dormant until spring. Evergreen daylilies grow throughout the year and do not form compact buds under either short day-length or low temperature. When exposed to freezing temperature, the loaves die, as does the entire plant in many instances. Semi-evergreen cultivars do not full y fit under either dormant or evergreen category, and growth behavior is commonly dictated by the prevailing environmental conditions. Many modern hybrids are in this class, resulting from the cross between evergreen and dormant types.

B. Inflorescences and Flowers

Daylily stems gives rise to 1 to 3 flower inflorescences per year. The inflorescences may have either an apical or axillary origin. The time of floral initiation varies greatly in daylilies. Depending on the cultivar, inflorescences can be initiated from July through December (Arisumi and Frazier 1968). One initiated, the inflorescence development is arrested until the next growing season in both evergreen and dormant types. Certain cultivars, commonly referred to as reblooming, can initiate and elongate more than one inflorescence in succession during a single growing season. 3. DAYLILY: BOTANY, PROPAGATION, BREEDING 201

The daylily inflorescence is a bostryx or modified cyme so that the right- or left-hand branch is the more vigorous than the apex (Voth et al. 1968). The inflorescences vary in length from 4 cm (H. darrowiana) to 200 cm (H. altissima); and can be erect, arched, or bowed down toward the ground under weight of their bloom, as is in case of H. multiflora. Not all species have branched inflorescences. In H. nano, the unbranched inflorescences carry a solitary bloom. In addition, the shape and arrangement of bracts on the inflorescence can be useful in identifying species. For example, the bracts in H. iniddendorffii are broad, oval shaped, and overlap each other (Hu 1968b). As each flower lasts only for a day, flower bud count is very important in daylily for prolonged bloom. Most rlaylily cultivars bloom for two to four weeks. Some cultivars, commonly known as bud builders, have an indeterminant inflorescence that continues to produce flower buds throughout the season. Dalilies are monoclinous plants (i.e., both pistils and stamens occur within the same flower). Flower parts consist of six segments: the inner three segments are petals while the outer three segments are sepals. The throat where the flower meets the stem is often a different color from the rest of the bloom. Protruding from the throat are six stamens terminating in anthers. Daylily pollen is normally brown in color but may also be reddish or yellow. In the center of stamens, the pistil is noticeably longer than the filaments and consists of a stigma that is connected through the style to the ovary within the perianth tube. The perianth is tubular from the ovary to the point where stamens are attached. The stigma itself consists of three small bulbous thickenings that exude a sticky substance during the optimum period of pollination. After pollination, the pollen tubes grow rapidly down the style, reaching the micropyle in 5 to 8 hr (Stout and Chandler 1933; Arisumi 1962). Pollen tube growth is then arrested for about 8 hr after which growth continues. Fertilization occurs 36 to 48 hr after pollination. After fertilization, seed capsule pods form that are either round or elongated elliptical and contain six ribs that open iii pairs. Capsules contain three layers of black seeds that are either round or elliptical with a small raised point. Capsules typical ripen in about 50 days after pollination. The flowers of daylily cultivars flowers come in several colors, color patterns, and forms. The colors range in shades and color patterns differ in intensity or type within or between sepal and petal. There are also wide variations in flower size, and forms may vary from informal to circular or triangular. The flower and other characteristics are covered in more detail in Table 3.3. S. K. GULIA. B. P. SlNli, J . CARTER, AND R. J. (;RIESBAcH

Table 3.3. List of major traits and their types taken into consideration tor daylily hybridization. \lajor traits Types Near-white, light yellow, strong yellow. Orange, upper, peach or melon, flesh tones, brown, use-pink, rose-red, pale red, (feel) red, dark irohogany red (almost black), purple and ,duolute black. Fluwui ilo,st hg alld dotting Plain color, dotting, diamond dusting and gold dusting F' lower color distribution Self-colored, blended, polychrome. bitone, reverse bitone, bicolor and reverse bicolor lower patterning Simple, eyed, banded, halo, watermark, edged tipped and picotee Flower throat color Yellow, orange, and green ['lower throat size Small, large and dilated Flower shape Side view: flat, trumpet, flaring, recurved and double. Front View: circular, triangular, star-shaped. spider, orchid-shaped and informal 1" lox or Shape of Segm en t Rounded, pointed, pinched and twisted Flower edges Tailored ribbed and ruffled Flower texture Rippled or ribbed, smooth or waxy Flower size Miniature (<7.5 cm). small 97.5-11.5). large (11.5-17.5 cm) and giant (>17.5) Flowering habit Diurnal (day blooming), extended blooming and nocturnal (night blooming) Flowering season Extra earl y , early, earl y—midseason, niidseason, late midseason, late, very late Growth habit Branching habit: dwarf (<30cm). small (30-50 cm), medium (51-80 cm) and tall (>80 cm) Leaf forms Overwintering: deciduous, evergreen and semi-evergreen Ploidy level Diploid. triploid and tetraploid Disease Resistance or susceptible to rust Source: Erhardt 1992.

Daylilies complete their reproductive cycle in a single day. Each flower on a daylily opens in the morning and by late afternoon under- goes rapid senescence. Unlike many flowers, ethylene is not involved in the senescence process in daylily (Lukaszewski and Reid 1989: Lay-Yee et al. 1992). Daylily petals undergo a series of chemical changes before and after the opening of bloom (Guerrero et al. 1998; Panavas et al. 1998; Stephenson and Rubinstein 1998). Upon opening, W

3. DAYLILY: BOTANY, PROPAGATION, BREEDING 203

fructan hydrolysis results in an increase in the osmolatity (Bieleski 1993). This increase results in petal and sepal expansion. Specific activities of cellulase and pectin methylesterase are highest before flower opening, and specific activities of polygalacturonase and beta- galactosidase increase after flower opening. In addition, the activities of proteinases (Stephenson and Rubinstein 1998), RNAses (Panavas et al. 1998), and DNAses (Panavas et al. 1999, 2000) increase almost simultaneously just prior to, or along with, flower opening. Senescence in daylily is characterized by changes in lipid metabo- lism (Bieleski and Reid 1992) and phloem export of carbohydrates (Bieleski 1995). In daylily, a cDNA for a putative cysteine proteinase has been cloned (Valpuesta et al. 1995; Guerrero et al. 1998), and Rubinstein's (Panavas et al. 1998, 1999, 2000) group has cloned six additional cflNAs designated as Dsa (daylily senescence associated). One gene (Dsa6), a putative Si-type nuclease, is expressed only in petals and the level of its message increases as the flower opens (Panavas et al. 1999).

IV. HORTICULTURE

A. Propagation Daylily propagation and culture practices have been extensively reviewed (Benzinger 1968; IJunwell 1996, 1998, 2000; Black 2003; Garber 2004; Latimer 2004). Daylilies are commercially propagated asexually by dividing the crown. Seed propagation is used for breeding (Munson 1989; Dunwell 1998). Seeds of deciduous daylilies require cold stratified at 0 to 7CC for 6 to 8 weeks to germinate: the seeds of evergreen daylilies do not require cold treatment (Grieshach and Voth 1957). It usually requires two years to flower from seed. After several years of growth, daylily plants are composed of several crowns that are interconnected. These large plants can be propagated asexually by separating the crowns into individual plants. The annual rate of new crown development is genotype dependant and varies from 1:3 to 1:25, averaging 8:1 (Apps 1995; Dunwell et al. 1995). Thus, it often can take 10 years or more to have adequate number of plants to meet market demand of a newly released cultivar (Dunwell 1998). There are other methods for asexual propagation. For example, a single crown can be cut into several pieces, which usually then develop into new growing points (Erhardt 1992). In addition, small 204 S. K. CUrIA, B. P. SINGH, J. CARTER, AND R. J. GRIESBACH

shoots may develop from buds on the inflorescence. These shoots can be easily removed and rooted into the soil to form new plants. Fully developed proliferations usually take 10 to 30 days to root well, and these plants usually flower within 12 to 15 months (Dunwell et at. 1995: Dunwell 1994 Dunwell (1998) obtained 14 proliferations from a single 'Lisa My Joy' plant that had four inflorescences. The application of growth regulators such as benzyladenine (BA), benylarnino purine (BAP), indoleacetic acid (IAA), and cycocel have been used both to increase the number of shoots that developed from a single bud on the inflorescence and to induce dormant buds to develop into shoots (Pickles 1997; Zurles 2002; Leclere et al. 2006). In vitro micropropagation can accelerate vegetative propagation. Daylilies can be micropropagated from young inflorescences (Meyer 1976; Pounders and Garton 1996), flower petals (Heuser and Apps 1976), ovaries (Krikorian and Kann 1980, 2002, 2003), suspension culture cells (Krikorian et al. 1981b; Smith and Krikorian 1991), isolated protoplasts (Fitter and Krikorian 1981; Ling and Sauve 1995; Aziz et al. 2003), anther filaments and immature seed embryos (Gulia and Carter 2007). Recently a liquid hioreactor system was developed for very large-scale propagation (Adelberg et al. 2007). Like most plants, daylily ability to regenerate from tissue culture is dependent on the genotype and source of the explant (Cheng et al. 2004). However, it is not dependent, as some believe, on the ploidy level of the plant (Adelberg et al. 2007). There are reports of successful regeneration of aseptically cultivated plantlets of Hemerocallis 'Autumn Blaze' in space (Levine and Krikorian 1992) aboard the shuttle Discovery during a 5-day mission within NASA's Plant Growth Unit (PGU) apparatus. But in another space experiment (a 132-day experiment on the space station Mir), daylily plants from embryogenic cell cultures produced poor growth and nuclear abnormalities (Krikorian 1999), Pioneering work of Krikorian and Kann (1979) with daylily cells demonstrated totipotency (i.e., the potential to grow new plants from a variety of aseptically cultured tissues and cells). Further work by Ling and Sauve (1995) resulted in regenerated daylily plants from protoplast-derived calli. The protoplasts underwent sustained division to produce multicellular colonies on MS medium supplemented with 0.5 mg/L naphthalene acetic acid (NAA) and 0.5 rng/L BA. Efficient regeneration from adventitious shoots can occur on clusters after subculture (Chen et al. 2005). Paclobutrazol and sucrose levels in the media significantly affected starch accumulation, growth value, and dry weight percenta ge of liqu id-cultured nieristemati c clusters. The p

3. DAYLILY: BOTANY, PROPAGATION. BREEDING 205

use of liquid shake cultures for mass proliferation of meristematic clusters followed by regeneration of adventitious shoots on semisolid agar culture could be an efficient system for large-scale micropropagation of daylily (Chen et al. 2005).

B. Pests and Diseases Diseases and insects are generally not a serious problem on daylily. Before onset of rust problem, da ylilies were considered almost disease and pest free (William-Woodward and Buck 2002). Daylily rust, caused by Puccinia hemerocallidis, was first reported in the United States during 2000 concurrently in Georgia and in Tennessee (William- Woodward et al. 2001; Windham et al. 2004). By late 2001, daylily rust was identified in 30 states, and has become a problem throughout United States (I-lernandez et al. 2002; Sakhanokho et al, 20041)). Mueller et al. (2003a, b) and Li et al. (2005) studied rust resistance and classified daylily cultivars into resistant and susceptible groups. Daylily rust has separate asexual and sexual life cycles. In the asexual life cycle, urediospores land on living plant tissue, germinate, and form mycelium within the leaf. Eventually the mycelium forms a mass of urediospores that infect new tissue and result in a buildup of the disease during the growing season. Conditions conducive to spore germination and growth are long periods of leaf wetness; temperatures between 15' and 30°C; and a high relative humidity between of 75% and 80% (Mueller and Buck 2003). At the end of the growing season, the myceliuin forms teliospores instead of urediospores. The teliospores lie dormant on dead leaves during the winter. In the spring, the teliospores germinate to produce basidiospores, which do not infect daylily but infect the alternate host Patrinia. In Patrinic,, the basidiospores form pycnia in which the sexual stage of the life cycle occurs. The sexual stage of the life cycle results in aeciospores, which infect daylily and start the asexual urediospore stage of the life cycle over again (Hernandez et al, 2002). Even though two spore types, urediospores and teliospores, are detected on daylilies within the United States, no one has yet discovered infected Patrinia plants (Hernandez et al. 2002). In addition, basidiospores have not been detected. Since it appears that daylily rust can not utilize the U.S. native species of Patrinia, it is hoped that the disease will not be a significant problem in colder areas of the United States. However, daylily rust has successfully over- wintered in the warmer areas of the United States (USDA Zone 7). In warm climates, teliospores are not required for winter survival since 206 S. K. GIJIJA. B. P. SING] 1. J. CARTER. AND R. J . GRIESBACH the asexual urediospore stage can continue through the winter. Some people are also concerned that daylily rust could persist in areas where there is a protective covering of deep and continuous snow throughout the winter or perhaps under a heavy winter mulch. Research is continuing into the biology and winter survival of this disease. Various studies also have been conducted to study• effects of application of various fungicides (Buck and Williams -Woodwards 2003; Mueller et al. 2005) and environmental factors such as light, temperature, and leaf wetness (Mueller and Buck 2003) on rust development. An interesting study by Reilly et al. (2005) has shown a correlation between rust susceptibility and nickel. Not only did nickel spray treatment (200 ppm) prevent the infection of clean plants, it also prevented the infection of new leaves formed on diseased plants. Along with rust, daylily plant can also be attacked by diseases such as leaf streak (A ureobasidium micro stictum), root-knot nematode (Meliodgyne incognita), soft rot (Erwinia carotovora), and insect pests such as flower thrips (Frankliniella tritici), two spotted spider mites (Tetranychus spp.), aphids and bugs (Lopidea confluenta), slugs and snails (Spencer 1972, 1973). Recently Armillaria root rot in daylily was reported (Schnabel et al. 2005) in South Carolina. Armillaria root rot causes stunting and necrosis of leaves at the tip, thereby lowering the esthetic value of the plant.

C. Cultivar Registration and Awards The American Hemerocallis Society (AHS) is the official Hemorocallis registrar (Gretchen Baxter, Registrar, American Hemerocallis Society, P0 Box 9887, Greensboro, NC 27429) and has a searchable electronic version their cultivar registration database call Day 'Dream' (Daylily Registry Electronic Access Modules) using FilemakerPro I NI software (www.daylilies.org). As of 2007, over 58,400 cultivars have been registered. Gosukonda et al. (2005) developed artificial neural networks (ANNs) to predict daylily hybrid patterns from known characteristics of parents used in hybridization. The traits included were height, diameter, foliage, blooming habit, ploidv, and blooming sequence in a cultivar analysis based on 230 genotypes. The results from prediction plots indicated a better accuracy from regression model than ANN models suggesting the need for more data sets within the domain of training of ANN for it to predict hybrid patterns. Thus, there is clear need to develop relational database with data-mining and 3. DAYLILY: BOTANY, PROPAGATION, BREEDING 207

data-visualization capability to provide a unified information resource to breeders. The American Hemerocallis Society also has a judging system that grants three different dultivar awards (Award of Merit, Honorable Mention and Junior Citations) based on a defined set of standards (Table 3.3). As of 2007, 4,091 cultivars have been awarded (www.daylilies.org/AllAHSAwards06.pdf). In 1985, the All-American Daylily Selection Council (AADSC) was organized to administer a network of test sites throughout North America to evaluate commercially available cultivars for garden performance (www.allamericandaylilies.com). To date, nearly 6,000 cultivars have been evaluated. Daylilies earning the AADSC's All- American" designation show superior performance across at least five USDA hardiness zones.

V. GENETICS

A. Genome and Ploidy Level Most Hemerocallis species are diploid with Ii pairs of chromosomes (Takenaka 1929; Stout 1932; Brennan 1992). Tomkins (2003, 2004) estimated the genome size at 4408 Mb (rnegahase pairs), which is comparatively smaller than barley, oats, wheat, and onion but larger than rice, sorghum, maize, and sugarcane (Fig. 3.2). Several taxa with the H. fulva species complex (H. fulva 'Europa', H. fulva 'Kwanso', H. fulva var. paucijiora Hotta & Matsuoka, and H. fulva var. maculate Baroni) are not diploid but naturally occurring triploids (2n = 3x = 33) (Stout 1932; Chandler 1940). Since there are no known tetraploid forms of any of the species, these triploid species did not arise from tetraploid/diploid crosses, Mostly likely they originated from an unreduced egg cell. Arisumi (1970) roughly estimated that the frequency of unreduced egg cells was 1 in 15,000. He did not find a single unreduced pollen grain. In some species, triploids can be created easily through tetraploid/ diploid crosses. However, in daylily, Arisumi (1973) obtained only 29 triploid seedlings in 1,607 diploid-tetraploid pollinations. The frequency of success was doubled when the tetraploid parent was female. There have been a number of reports that amateur breeders have obtained "triploid" plants from tetraploid/diploid crosses; however, most of these "triploids" turn out to be diploid. The tetraploid parent used in these crosses usually was derived from a colchicine-induced tetraploid chimera (R.A. Griesbach pers. commun.).

208 S. K. GULIA, B. P. SINGH, J. CARTER, AND R. J. GRIESBACH

183)00 17,000 16,000 15,00(1 14.000 13.0)))) 'a 12.)))))) (1.000 10.00)) •9,000 8.00)) 7.000 6,1(0)) 5,000 4,000 3,000 2.000 1.000 ------.I1II1 t) C t) U >'. >, C - C.- -- C U C cc

Plant Species

Fig. 3.2. Histogram showing the genome size of various plant species in comparison to daylily genome. (Source: Database of genome sizes (DOGS) online available at www,cbs. dtu.dk/databases/DQGS/jndex,htrnl)

Triploid daylilies are not very fertile due to the formation of multivalent chromosome pairing during meiosis. In H. fulva var. Kwanso, the 11 trivalents formed during meiotic prophase I resulted in 33 univalents during metaphase I and irregular tetrads containing various numbers of nonviable pollen grains (Dark 1932). Even though daylily triploids are not very fertile, they can be used in breeding. Stout (1926) obtained 1% viable seed using H. fulva 'Europa' as a female parent and 15% viable seed when using 'Europa' as the male parent. The ability of colchicine to induce polyploidy was discovered in 1937 (Blakeslee and Avery 1937) and used in daylily during the 1940s to create tetraploids. The difference between tetraploid and diploid forms of the same daylily is striking. Inmost instances, the tetraploid flower is larger, heavier in substance, and more richly colored (Stamile 1990; Kehr 1996; Petit and Callaway 2000; Sakhanokho et al. 2003). In 1947, the first colchicine-induced tetraploid daylily ('Brilliant Glow') was produced by Robert Schreiner, a student at the University of Minnesota, by treating the diploid 'Cessida'. In 1948, Quinn Buck at 3. DAYLILY: BOTANY, PROPAGATION, BREEDING 209

the University of California reported flowering the tetraploid forms of 'Soudan' and 'Kanapaha'. A year later, Hamilton Traub of the U.S. Department of Agriculture in Maryland flowered the tetraploid form of 'Mayor Starsynski' named 'Tetra Starzynski' (Traub 1959 and 1960). Not all coichicine-induced tetraploid daylilies produce tetraploid offspring. The daylily meristem has three different apical cell layers (L-I, L-II, L-III). The L-I layer is responsible for forming the epidermis of all organs and forms the mesophyll tissue along the leaf margin. The pollen and seeds originate from the L-II, as do, the entire mesophyll tissue of the petals and sepals and the mesophyll tissue in the outer region of the leaf next to that formed from the L-I. The L-III forms the mesophyll tissue in central region of leaf and does not contribute to any floral tissue. Colchicine treatment of daylily plants nearly always results in chimeras rather than in pure polyploids (Arisumi 1964), There are three types of chimeras: mericlinal, periclinal, and sectorial. In mericlinal chimeras, the tetraploid tissue occurs in cells along the side of the meristem and results in tetraploid tissue only on one side of the plant. Mericlinal chimeras are unstable and usually revert back to pure diploidy. In periclinal chimeras, the tetraploid tissue occurs in one (or more) of the meristem layers (L-I, L-II, or L-II), in a hand-in- glove configuration. Periclinal chimeras are relatively stable. In sectorial chimeras, the tetraploid tissue occurs as a solid section through all apical layers on only one side of the meristem. Thus, cell division products of the tetraploid cells give rise to a section of tetraploid tissue. Sectorial chimeras are relatively stable. When using colchicine-treated plants in breeding, it very important to identify what type of chimera they are and the ploidy of their L-II layer. Besides chromosome counts, stomata size, pollen size (Arisumi 1965), and flow cytometry (Saito et al. 2003) have been used for determining ploidy. The drawback in using stomata size is that it measures the ploidy level of L-1 tissue and not L-II tissue. The first tetraploids were not as fertile as diploids (Peck and Peck 1969), due the production of multivalents during meiosis. Artificially chromosome-doubled plants are autotetraploid and form quadrava- lents during meiosis that can lead to abnormal meiosis (Fig. 3.3). For example in 'Crestwood Ann', numerous multivalents during meiosis resulted in lagggers and bridges. Over time, more fertile tetraplopids were selected in breeding. These fertile plants had a normal meiosis. This is similar to what was found over time in maize (Gilles and Randolph 1951). Specific genes have been described in several species that prevent multivalent formation (Jackson and Casey 1982). Similar

210 S. K. GULIA, B. P. SINGH, J. CARTER, AND R. J. GRIESBACH I!

Fig. 3.3. Meiosis in daylily: (A) Crestwood Ann' showing metaphase 1: (B) 'Crestwood Ann' showing anaphase I with abnormal cell-plate formation; (C) 'Crestwood Ann' showing anaphase 1 with laggers forming micopollen grain; and (D) 'Crestwood Ann' showing anaphase I with unequal multivalent separation forming bridge. genes most likely played a role in developing more fertile tetraploid daylilies. Most modern tetraploid cultivars are now as fertile as their diploids. The first major tetraploid breeding program was started in 1955 by Robert A. Griesbach at DePaul University in Chicago, Illinois, and Orville Fay, a retired chemist. They developed a new method of coichicine treatment utilizing germinating seedlings. The seed treatment was easier and more efficient than the whole-plant treatments used by Schreiner, Buck, and Traub (Traub 1959, 1960). Using the seed treatment, one could treat a larger number of plants in the same amount of time. In addition, the seed treatment resulted in a higher frequency of tetraploidy (Griesbach et al. 1963). Besides publishing the treatment method, Griesbach held numerous training sessions to teach the procedure to both commercial and amateur breeders. F

3. DAYLILY: BOTANY, PROPAGATION, BREEDING 211

Table 3.4. Point scale standards for judging daylilies. Characteristic Points Complete plant 30 Garden value (10 pt) Vigor (10 pt) Performance (10 pt) Foliage 10 Inflorescence 20 Height (10 pt) Branching/bud count (10 pt) Flower 30 Durability/fragrance (10 pt) Color (10 pt) Form (10 pt) Distinction 10 TOTAL 100 Source: American Hemerocallis Society, 2008.

In 1961, Fay and Griesbach released four tetraploids: 'Crestwood Ann', 'Crestwood Bicolor', 'Crestwod Evening', and 'Crestwood Lucy'. Unlike the previously released tetraploid cultivars, the Crestwood cultivars were reasonably priced and widely distributed. The wide distribution of these cultivars, coupled with Griesbach's training sessions, resulted in a new wave of daylily breeding (Traub 1973). Through the years, refinements in the colchicine treatment procedure have occurred (Arisumi 1964, 1972; Buck 1969; Warner 1969; Chen and Goeden-Kallemeyn 1979; Barr 1990; Brennan and King 2003; Sakhanokho et al. 2004a). One of the refinements by Toru Arimsu of the U.S. Department of Agriculture in Maryland involved treating the exposed vegetative meristem (Arisumi 1964). This very successful method for converting diploid cultivars into tetraploid clones was used by many amateur breeders during the 1960s and 1970s to expand the tetraploid gene pool (Traub 1973). A number of studies have been carried out to determine genetic diversity among daylily populations using isozymes and allozymes (Kang et al. 1998; Kang and Chung 2000), AFLP (Tomkins et al. 2001), and chioroplast DNA (Noguchi et al. 2004) markers. Isozymes studies by Kang and Chung (2000) revealed high level of allozyme variation within 30 populations of five Hemerocallis species from Korea and low level of allozyme divergence within species. Tomkins et al. (2001) conducted diversity analysis of 19 primary genotypes and 100 cultivars of daylily from different time periods 212 S. K. (221\. IL I. 1;H. J. (\kTkR. \\I) K J. (KIIILLFI using AFLP markers. They observed that there was a slight decrease over time in the genetic diversity of diploid cultivars that were released between 1940 and 1964. From 1965 to 1980, the genetic diversity remained unchanged. Interestingly, the genetic diversity of 40 tetraploid cultivars released froni 1980 to 1998 was slightly lower (Nei's similarity coefficient of 0.850) than that of 21 diploid cultivars released during the same period (0.814). The genetic diversity of the diploid cultivars released during this time period was significant lower than that found in the wild species (0.762). These data suggest that there is need to further increase both the tetraploid and diploid gene pools.

B. Flower Color Inheritance Flower color in daylily is the result of three pigments: chlorophylls, carotenoids, and flavonoids. Flavonoids can be further divided into copigments (colorless) and anthocyanins (colored). Chlorophylls are located within chioroplasts in the cell cytoplasm. Carotenoids are contained within chromoplasts, whereas flavonoid and betalains are located within the cell vacuoles. The yellow through orange colors of flowers typically are due to the carotenoid pigments, whereas blue to red colors typically are attributed to anthocyanins. Chlorophylls are responsible for green color. Each type of pigment is the result of a different sequence of biochemical reactions. The production of each pigment is independent of the other pigments. In most cases, a defect in the flavonoid pathway has no effect on the carotenoid and chlorophyll pathways and vice versa (Griesbach 1984, 2005). Most daylily flowers derive their color from more than one pigment source (Griesbach and Batdorf 1995). It is very striking to compare the flower color of modern cultivars with that of the species. Modern cultivars come in a wide range of colors, from purple through red and yellow through orange; the flower color of nearly all of the species, however, is limited to yellow through orange. Two taxa have unique colors: Hemerocallis fulva fm. rosea has rose- colored flowers and H. fulva fm. disticha has mahogany-colored flowers. The orange flower color of the wild type H. fulva fm. fulva (Munsell 7.5R 7/14) is the result of a single anthocyanin (cyanidin-3-rutinoside) and two carotenoids (zeaxanthin and lutein) (Asen and Arisumi 1968; Griesbach and Batdorf 1995). The novel mahogany flower color of H. fulva fm. disticha (RHS 17113) is the result of a mutation in the flavonoid pathway leading to delphinidin-3-rutinoside instead of PF

3. DAYLILY: BOTANY, PROPAGATION, BREEDING 213

cyanidin-3-rutinoside. Similar to H. fulva frn. fulva, I-!. fulva fin. disticha contains the two carotenoids zeaxanthin and lutein. The rose flower color of H.fulva fm. rosea (Munsell 2.5YR 5/10) is the result of mutation that reduces the concentration of the caroteinoid pigments while maintaining the concentration of anthocyanin cyanid i n-3 -rutinoside, During the 1960s, a group of breeders (Fay, Griesbach, and Peck) determined the inheritance of a mutation that reduced the concentration of anthocyanin pigments while maintaining the concentration of caroteinoid pigments. They gave this recessive mutation the name melon, In 1934, the New York Botanical Garden released three daylily cultivars with new red flower colors that seen in the wild species (Stout 1942). The cultivar 'Theron' with dark red flowers was derived from H. x aurantiaca, H. fulva, and H. flava. The cultivar 'Red Bird' with scarlet-red flowers was derived from intrabreeding several unique forms of H. fulva from Chengtu and Kuling, China, with orange-scarlet flowers instead of the typical orange color. The cultivar 'Rosalind' with rose-red flowers was derived from intrabreeding three different H. fulva fm. rosea clones. Theron', 'Red Bird', and 'Rosalind' led to the development of modern true red, pink, and purple flowered cultivars.

C. Biotechnology The tissue culture techniques previously described can be used to produce transgenic daylily plants. Mutants can arise through the tissue culture process. These mutations are called somaclonal (Larkin and Scowcroft 1981). Somaclonal mutations can arise from mutagenic chemicals used in the tissue culture process. In addition, the tissue culture process allows naturally occurring mutant cells to develop into whole plants. For example, a leaf cell containing a mutation in a flower color gene would express that mutation only if it were regenerated into a whole plant. Hemerocallis 'Yellow Tinkerbelle' is a somaclonal mutation of H. 'Eenie Weenie' that has a more dwarf growth habit (Griesbach 1989). The frequency of somaclonal mutations is reported to be very low in daylily (Krikorian et al. 1981a; Griesbach 1989). Griesbach (1990) has suggested delaying the shoot formation, use of undifferentiated tumorlike callus cells or older callus, and use of auxins as ways of increasing the frequency of sornaclonal variants. Regeneration protocols and genetic transformation of daylily (Hemer ocallis spp. 'Stella de Oro') by particle bombardment have been achieved (Aziz et al. 2003). Callus cultures initiated from ovules were bombarded with gold particles coated with plasmid-harboring Bastaj 214 S. K. GULIA. B. P. SINGH, J. CARTER. AND R. J . GRIESBACH resistance gene. Resulting putative transgenic calli were selected after 2 weeks, and surviving calli regenerated shoots after 2 months. Polymerase chain reaction and Southern blotting were used to confirm independent transformation events. Genetic engineering could prove useful to introduce new traits that do not exits in the gene pool.

VI, CONCLUSION

Daylily is an important ornamental crop that also has culinary and medicinal uses. There is scope for further expansion of its use in landscaping because the plant is drought resistant and requires low maintenance. However, due to asexual mode of propagation in daylily through slow-dividing crowns, it has been difficult to meet the market demand for choice new cultivars. The use of tissue culture on commercial scale to accelerate propagation rate may hold the key to solving this problem. It should be kept in mind that separate tissue culture protocols may be needed for individual cultivars. Until now, daylily breeding has been carried out mostly by amateur breeders. Objectives of these breeding efforts were to produce dultivars differing in flower characteristics: notably color, shape, and form of flowers. A large number of diploid and tetraploid daylily cultivars were produced for these purposes. Considering the market potential for daylily, now there is need for genetic studies and widening of gene pool to incorporate value-added traits in new daylily cultivars. Ornamental use of daylily as cut flowers has potential, provided flowers longevity is extended to multiple days. The genes for this purpose are available in other species, but their introgression into daylily is awaited. The lucrative markets of daylily for food and medicine are essentially untapped. In summary, daylily is an important land inflorescence plant that holds promise of considerable expanded use for this and other purposes provided its propagation problems are solved and certain needed traits are incorporated into the crop.

VII. LITERATURE CITED

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