HORTSCIENCE 39(6):1220–1223. 2004. Hybridization. In winter and spring months, photoperiod was increased to 16 h using night-interruption lighting (with high pressure Inheritance of Flower Color in sodium lamps) from 2200 to 0200 hr to induce fl owering. Hybridizations were performed on Anagallis monelli L. emasculated, unopened buds and pedicels were tagged for identifi cation. Approximately three 1 Rosanna Freyre weeks were allowed for fruit formation, and Plant Biology, University of New Hampshire, G36 Spaulding Hall, Durham, then watering was stopped to dry the plants NH 03824 and fruit. Fruit were harvested when brown and brittle on the outside, the placenta dry, Robert J. Griesbach and seeds a dark brown or black color. F1 and Floral and Nursery Plant Research, U.S. National Arboretum, U.S. Dept. F2 seeds were sown in seed trays containing of Agriculture, Agricultural Research Service, BARC-West, Building 010A, 360 Scotts coir, covered lightly with media and placed on a bench with intermittent mist. Beltsville, MD 20705 Trays were removed from the mist upon emer- Additional index words. Primulaceae, anthocyanins, fl avonoids gence of the fi rst seedlings. Initially they were transplanted into small cell packs and later into Abstract. Plants of Anagallis monelli in their native habitat or in cultivation have either 15- or 25-cm pots. blue or orange fl owers. Clonally propagated cultivars, seed obtained from commercial Color and pH determination. Flower sources and the resulting plants were grown in a greenhouse at the University of New Hampshire. F2 progeny obtained from hybridization between blue- and orange-fl owered plants had blue, orange or red fl owers. There were no signifi cant differences in petal pH of orange-, blue-, and red-fl owered plants that could explain the differences in fl ower color. Anthocyanidins were characterized by high-performance liquid chromatography. Results indicated that blue color was due to malvidin, orange to pelargonidin, and red to delphinidin. Based on our segregation data, we propose a three-gene model to explain fl ower color inheritance in this species. The genus Anagallis is in the family patents 10/721,990 and 10/721,991, respec- Primulaceae, although recent phylogenetic tively) were released from the ornamental studies based on DNA sequence data from breeding program at the University of New three chloroplast genes and morphology have hampshire (UNH) in 2002. placed it in the family Myrsinaceae (Källersjö Harborne (1968) found the fl avonols quer- et al., 2000). There are about 28 species in the cetin and kaempferol in fl owers of A. arvensis genus Anagallis, mostly native to Europe, Asia, and A. linifolia, and cited research fi nding Africa and America (Clapham et al., 1987). 3- and 3,5-glycosides of malvidin, delphinidin Anagallis monelli L. (taxonomic synonyms A. and pelargonidin in different color forms in A. collina Schousboe, A. linifolia L.) is a short- arvensis. Ishikura (1981) identifi ed malvidin lived perennial with blue fl owers found in dry, 3-rhamnoside, luteolin, luteolin 7-glucoside open habitats in southwestern Europe (Tutin and quercetin 3-rhamnoside in blue-fl owered A. et al., 1972). Wild forms are diploid (2n = 20) arvensis. Elsherif (2000) isolated chalcone syn- (Talavera et al., 1997) and self-incompatible thase and fl avanone 3-hydroxylase from fl owers (Gibbs and Talavera, 2001; Talavera et al., of A. monelli ‘Skylover Blue’ and ‘Sunrise’, and 2001). A variant, diploid form of A. monelli reported fi nding only pelargonidin derivatives in with orange fl owers is found in southern Italy fl owers of ‘Sunrise’ and only malvidin deriva- and northern Africa (Freyre, unpublished data; tives in fl owers of ‘Skylover Blue’. Talavera, personal communication). We obtained A. monelli plants with a novel Cultivated forms of A. monelli (blue pimper- red fl ower color from hybridization between nel) are vigorous with large, deep blue fl owers blue- and orange-fl owered plants. The objectives and are used as annual bedding plants and for of this study were to determine the biochemi- hanging baskets. ‘Gentian Blue’ is a seed- cal basis of blue, orange and red fl ower colors propagated blue cultivar sold by Thompson and propose a genetic model for inheritance of & Morgan (Jackson, N.J.). Presently, this and fl ower color in A. monelli hybrids. several other commercial seed companies carry only unnamed cultivars of blue pimpernel. Materials and Methods Several vegetatively propagated cultivars are offered in the trade including blue-fl owered Plant material. Plants of A. monelli ‘Sun- ‘Skylover Blue’ and ‘Wildcat Blue’ and or- rise’ and seeds of ‘Gentian Blue’ were obtained ange-fl owered ‘Sunrise’ and ‘Wildcat Orange’. from commercial sources and grown at UNH. ‘Wildcat Blue’ and ‘Wildcat Orange’ (plant All plants were maintained in 25-cm baskets using 560 Scotts coir soiless medium (The Received for publication 29 Aug. 2003. Accepted Scotts Co., Marysville, Ohio) during cooler for publication 23 Dec. 2003. Scientifi c contribu- months or 360 Scotts coir medium in the tion 2198 from the New Hampshire Agricultural o o Experiment Station. We thank Thomas M. Davis summer, in a greenhouse with 21 C day/18 C for valuable discussions; Amy Douglas and Deborah night set points. Fertilization was constant with Schneider for assistance in greenhouse research; and a 20N–4.3P–16.7K fertilizer at a maximum of Kenneth Schroeder, Dennis Werner, and John Ham- 150 mg·L–1 N. To ensure healthy growth, the mond for critically reviewing the manuscript. growing medium pH was maintained between 1To whom reprint requests should be addressed; 5.7 to 6.3 and electroconductivity between 1.0 Fig. 1. Flowers of blue-, orange-, and red-fl owered e-mail [email protected]. and 2.0 mS·cm–1. Anagallis monelli. 1220 HORTSCIENCE VOL. 39(6) OCTOBER 2004 0080-Breed.indd80-Breed.indd 11220220 99/20/04/20/04 33:44:57:44:57 PPMM color was determined using Munsell notation ‘Sunrise’ has low pollen and ovule fertility din, while orange (Munsell 1.3YR 5.6/16) and (Nickerson, 1946). The Munsell Book of Color and is diffi cult to use in breeding. After many red (Munsell 0.3R 4.8/17) colors were due to (Munsell Color Services, New Windsor, N.J.) attempts, two F1 seedlings of ‘Gentian Blue’ predominance of pelargonidin and delphinidin, was used instead of the Royal Horticultural x ‘Sunrise’ were obtained, both of which had respectively (Table 2). As expected, multiple Society’s Colour Charts because it is not pos- orange fl owers. Since the two F1 seedlings anthocyanins were found in the different fl ower sible to interpolate between color chips using were partially self-compatible, all F2 combina- color types, since nearly all of the anthocyanin the RHS Colour Charts. The pH of petal tissue tions (F1-Orange1 selfed, F1-Orange2 selfed, biosynthetic enzymes that have been studied was determined one day after fl ower opening. F1-Orange2 x F1-Orange1, and F1-Orange1 x will accept the different dihydroflavonol It was previously determined that the pH of an F1-Orange2) could be produced. None of these precursors as substrates (Fig. 2; Huitts et al., epidermal peel suspension accurately refl ected combinations resulted in large populations; 1994). Different anthocyanin profi les in fl owers the pH of a single cell (Griesbach, 1998; Stewart therefore, all the populations were pooled. A are the result of differences in relative Km’s for et al., 1975). Therefore, the upper epidermis total of 65 F2 plants were obtained, 28 of which the various substrates. When doing inheritance was stripped and the epidermal strips from were from F1-Orange2 selfed, 19 from F1-Or- studies, the predominant anthocyanin is used as a single fl ower were combined. Strips were ange1 selfed, and 18 from the F1-sib cross. In the phenotype (Griesbach et al., 1991). then ground into a suspension with distilled the F2 population, 55 plants had orange fl owers, It is generally assumed that delphinidin is water and the pH of the suspension measured 8 plants had blue fl owers, and 2 plants had red the pigment responsible for blue fl ower colors with a micro-pH meter (Sentron 501; Sentron, fl owers (Fig. 1). This segregation ratio (55:8:2) and cyanidin is the pigment responsible for red Inc., Federal Way, Washington D.C.). The pH fi t a three gene model (55:9:3) with a χ2 = fl ower colors (Forkmann, 1991). However, measurements were recorded as a mean of 10 0.593, p = 0.74. Additional crosses were made there are exceptions to this generalization. replications, each replicate representing the (F1-backcross, F3, etc.), but populations’ sizes For example, the blue color in Meconopsis pooled tissue collected from a single fl ower. were too small for genetic analysis. However, grandis Prain. is due to cyanidin (Takeda et al., Anthocyanidin analysis. Fresh fl owers these crosses resulted in additional red-fl ow- 1996), while the red color in Petunia exserta were ground in 1% (v/v) HCl in methanol. ered seedlings (Red3, Red4, Red5) that were Stehmann is due to delphinidin (Ando et al., The extract was fi ltered and reduced to dryness used in further biochemical analysis. 2000). Under acidic conditions it is possible under reduced pressure at 40 oC. The residue A sample of fi ve to seven plants for each for delphinidin to appear red (Asen, 1976). was dissolved in 1% (v/v) HCl in methanol color was used for biochemical analysis. Re- The vacuole pH can affect the petal color by and clarifi ed by centrifugation at 100,000 gn sults of comparative UV spectrophotometry modifying the physical interaction between for 2 min. with known standards identifi ed three different the electrons in the pigments (Brouillard, The anthocyanins were characterized by anthocyanidins: pelargonidin, delphinidin and 1998).