Reproductive Behavior of Diploid and Allotetraploid Rhododendron L. Fragrant Affinity

Wide hybridization can lead to recombination of diverse traits and creation of unique phenotypes, but the resultant hybrids are often sterile as is the case with the intersubgeneric hybrid Rhododendron L. Fragrant Affinity . Sterility in wide hybrids can either be genic or chromosomal; the latter may be overcome by induction of polyploidy, which can restore chromosome homology and fertility. Cytologic studies of Fragrant Affinity appear to support the presence of bridges between bivalents in metaphase I and laggard chromosomes in anaphase I. In the current study, an allotetraploid form of R. Fragrant Affinity was developed using oryzalin (4-(dipropylamino)-3, 5-dinitro-benzenesulfonamide) as a mitotic inhibitor and chromosomal doubling agent. Genome sizes (2C) were determined using flow cytometry and found to be 1.6 and 3.2 pg for the diploid and allotetraploid, respectively. Pollen viability, determined by staining and germination tests, was 4% and 0%, respectively, for the diploid and 68% and 45%, respectively, for the allotetraploid. No seeds were produced when the diploid R. Fragrant Affinity was used as a pistillate parent when pollinated with pollen from viable diploid and tetraploid parents. The allotetraploid produced viable seeds and seedlings when pollinated with pollen from either diploid or tetraploid parents, including self-pollination, demonstrating restored male and female fertility. Rhododendrons and azaleas (Rhododendron L.) are among the most widely grown ornamental plants in the world. There are over 1000 species recognized in eight subgenera (Chamberlain et al., 1996). Species within subgenera readily hybridize and have resulted in thousands of cultivars (Väinölä, 2000). Although intrasubgeneric hybridization is responsible for the majority of existing cultivars, species in different subgenera are sometimes capable of hybridizing. Azaleodendrons are hybrids between deciduous azaleas (subgenus Pentanthera (G.Don) Pojarkova) and nonscaly leaved rhododendrons (subgenus Hymenanthes (Blume) K.Koch) and constitute some of the oldest hybrids within the genus. The first interspecific hybrid rhododendron reported was Azaleoides resulting from a chance cross between Rhododendron periclymenoides (Michx.) Shinners and R. ponticum L. in London circa 1820 (Hillier Nurseries, 2002). Such hybrids have the potential to combine the fragrance of the deciduous azaleas with darker flower colors, larger flower size, and persistent foliage of evergreen rhododendrons. One such hybrid with breeding potential is Rhododendron Fragrant Affinity . Fragrant Affinity is an azaleodendron with semievergreen foliage, vigorous growth, good cold hardiness (–26 C), and fragrant, lavender flowers (personal observations). This intersubgeneric hybrid, believed to be a cross between R. ponticum and R. viscosum (L.) Torrey (Contreras, 2006), possesses unique attributes that are desirable for breeding and development of superior, cold-hardy, fragrant azaleodendrons. Unfortunately, like many other wide hybrids, it appears to be sterile. Hybrid sterility, referred to as chromosomal sterility or chromosomal hybrid sterility, is often the result of improper chromosome pairing during gametogenesis resulting from structural differences in parental chromosomes. This results in meiotic abnormalities such as univalents and lagging chromosomes; however, other mechanisms may also be involved in hybrid sterility (Lu and Bridgen, 1997). In a study using Alstroemeria aurea Graham · A. caryophyllaea Jacq., Lu and Bridgen (1997) determined that its sterility resulted from complex fertility/sterility-regulating mechanisms and was not simply the result of parental chromosome differences. In cases in which sterility is incited by improper chromosome pairing, doubling the chromosome complement (polyploidization) of sterile hybrids to produce allotetraploids provides a homolog for each chromosome to pair with during meiosis and can allow for the development of fertile gametes (Hadley and Openshaw, 1980; Lu and Bridgen, 1997; Stebbins, 1950; van Tuyl and De Jeu, 1997; Zadoo et al., 1975). Natural polyploids exist in the genus Rhododendron, including triploids (2n = 3x = 39), tetraploids (2n = 4x = 52), hexaploids (2n = 6x = 78), octaploids (2n = 8x = 104), and dodecaploids (2n = 12x = 156) (Ammal, 1950; Ammal et al., 1950). Artificial polyploid rhododendrons have also been developed to increase ornamental characteristics such as flower size and texture, extend time of flowering, produce more compact plants, and facilitate crosses not possible at the diploid level (Eiselein, 1994; Kehr, 1996a, 1996b; Pryor and Frazier, 1968; Tolstead and Glencoe, 1991; Väinölä, 2000). Polyploid rhododendrons have been induced with various chemical doubling agents, including colchicine (Kehr, 1996a) and oryzalin (Väinölä, 2000). The objective of this study was to evaluate fertility of diploid and allotetraploid forms of R. Fragrant Affinity and to determine if induced polyploidy is an effective method for restoring fertility in hybrids of distantly related rhododendrons. Materials and Methods Plant material. A single plant of Rhododendron Fragrant Affinity was received from Dr. August Kehr in 2000. Semihardwood cuttings were taken in late summer, treated with 5000 ppm K-IBA, set in 1 peat:1 perlite (by volume), and placed in a chamber with intermittent mist at a rate of 10 s every 10 min. After rooting, plants were grown in pine bark medium amended with 0.59 kg m dolomitic lime and 1.0 kg m micronutrient blend (Micromax; Scotts, Marysville, Ohio) under 50% shade. Plants were fertilized using 17N–7.4P–14.1K controlled-release fertilizer (Multicote; Vicksburg Chemical Co., Vicksburg, Mo.). Plants used in controlled crosses were container-grown with the same media and conditions. Plant material was maintained at the Mountain Horticultural Crops Research Station (MHCRS), Fletcher, N.C. Received for publication 5 July 2006. Accepted for publication 30 Aug. 2006. This research was funded, in part, by the American Rhododendron Society (ARS), Niagara Falls, N.Y., the North Carolina Association of Nurserymen (NCAN), Inc., Raleigh, N.C., and the North Carolina Agricultural Research Service (NCARS), Raleigh, N.C. From a thesis submitted by Ryan N. Contreras as partial fulfillment of the requirements for the M.S. degree. We thank Tom Eaker, Joel Mowrey, and Nathan Lynch for their technical assistance. Graduate Research Assistant. Current address: Department of Horticulture, University of Georgia, Athens, GA 30606-7273. Professor. Senior researcher. To whom reprint requests should be addressed; e-mail [email protected]. HORTSCIENCE VOL. 42(1) FEBRUARY 2007 31 JOBNAME: horts 42#1 2007 PAGE: 1 OUTPUT: December 26 22:37:34 2006 tsp/horts/131494/01723