Youping Sun

Date of Award


Level of Access Assigned by Author

Open-Access Dissertation

Degree Name

Doctor of Philosophy (PhD)


Plant Science


Donglin Zhang

Second Committee Member

Stephanie Burnett

Third Committee Member

Michael Day


Ilex glabra (L.) A.Gray (inkberry) is a native evergreen shrub with dark green foliage and compact habit. This shrub has gained popularity in the northern landscapes of the United States and more nursery growers would like to produce it. To better understand genetic relationships among inkberry cultivars and breed cold-hardy cultivars for northern nursery growers and landscape specialists, the following projects were conducted. A group of 48 inkberry accessions and two other Ilex species (Ilex crenata Thunb. and I. mutchagara Makino) were studied using amplified fragment length polymorphism (AFLP) markers. A total of 229 markers between 50 and 500 base pairs (bps) were produced from eight AFLP primer combinations. Eighty-seven percent of the markers were polymorphic. The markers for each primer-pair ranged from 22 to 45 and the genetic distance ranged from 0.001 to 0.349. Within the inkberry clade, 48 accessions were classified into six groups including, a wild species group and five cultivated groups, ‘Densa’, f. leucocarpa, ‘Pretty Girl’, ‘Shamrock’ and ‘Viridis’. Within each group, the legitimacy of named cultivars and distinguished clones were discussed based on both morphological and molecular data. Inkberry was hybridized with cold-hardy male Ilex verticillata (L.) A. Gray (common winterberry) and Ilex × meserveae S.Y. Hu (meserve holly) to increase the cold hardiness. Cross pollination of inkberry and its five cultivars with both male plants was carried out in a greenhouse. Inkberry ‘Chamzin’ and ‘Densa’ had higher compatibility with either common winterberry or meserve holly, while inkberry wild species and its cultivar ‘Compacta’ were less compatible, and ‘Nigra’ and ‘Shamrock’ were almost incompatible. The pollen germination in situ observed with fluorescence microscope also supported the above results. Pollen germination of common winterberry and meserve holly on the stigma of inkberry wild species and inkberry ‘Chamzin’, ‘Compacta’, ‘Densa’ was more than that on inkberry ‘Nigra’ and ‘Shamrock’. A dramatic reduction in the number of pollen tubes was observed as they grew along the style and into ovary. The percentage of pollen tubes reaching the ovary of inkberry wild species and inkberry ‘Chamzin’, ‘Compacta’, ‘Densa’ was higher than that of inkberry ‘Nigra’ and ‘Shamrock’. Most of their fruit set were aborted and the fully developed seeds were less than 54.2% (meserve holly) and 32.4% (common winterberry). Reproduction barriers, including the inhibition of pollen germination, pollen tube growth to the style and the ovary, and lack of fertilization, resulted in the cross incompatibility of inkberry with both cold hardy species. Further cross-pollination should consider the incompatibility of cultivar variations. Nodal segments containing one axillary bud (1-1.5 cm) of inkberry were established on a Murashige and Skoog (MS) medium without hormones. The sprouted shoots (~1.0 cm) were cultured on a MS medium supplemented with BAP, KIN or ZT at 2.3, 4.5, 9.1, or 18.2 µM. After 38 days, ZT and BAP induced multiple shoot formation with multiplication rates of 4-6, while the multiplication rate of KIN was less than 2. Shoots cultured on ZT grew significantly taller than those on BAP and KIN. The height of the longest shoots treated with ZT was 4.6 cm, 1.6-2.2 times greater than those treated with BAP or KIN. Shoots (~2 cm) were subcultured on ¼ strength MS (¼ MS) medium containing either IBA or NAA at 2.6, 5.1, or 10.3 µM. Adventitious roots formed in vitro after 2-4 weeks. IBA at 10.3 µM produced the best rooting (100%) compared to other treatments after 38 days of culture. The average number of roots per shoot for IBA was about 15, 1.6-3.1 times as many as that of other treatments. All rooted plantlets were successfully transplanted. Cold hardiness tests of inkberry cultivars were conducted in both field trials and laboratory tests. A total of 72% and 93% of plants survived for 2007 and 2008 planting, respectively. ‘Shamrock’ was the most cold-hardy cultivar; f. leucocarpa, ‘Viridis’, and ‘Nigra’ were the least cold-hardy cultivars; while ‘Compacta’, ‘Densa’, ‘Chamzin’, ‘Pretty Girl’, and wild species had intermediate cold hardiness. Based on controlled freezing test of inkberry cultivars, the REC50 value of inkberry cultivars ranged from -19 to -32 ?? for Jan. 2007 and -18 to -38 ?? for Jan. 2008. The cold hardiness rate from field trials was significantly correlated with the REC50 value from laboratory tests. Laboratory test could be used to reliably predict the cold tolerance of inkberry cultivars in the field.