Rudbeckia hirta ‘Plainview Farm’, a new multiple-layered ray flowered cultivar, shows potential for potted plant production. After years of seed germination, this specific flower morphological trait was still unstable from generation to generation. To maintain its unique features, leaf sections (0.25 cm~(2)) were cultured on Murashige and Skoog (MS) medium supplemented with either BA (0.5, 1.0, or 2.0 mg·L~(-1)), KIN (2.5, 5, or 10 mg·L~(-1)), or ZT (0.5, 1.0, or 2.0 mg·L~(-1))toinduce callus and microshoots. After cultivation for 33 days, all cytokinin treatments significantly induced callus and the callus size were 1.5- to-2.4-fold bigger than those withoutcytokinin. KIN at 2.5 mg·L~(-1) was the best treatment for callus induction and microshoot formation. Four microshoots per explant wereproduced at KIN of 2.5 mg·L~(-1). For rooting, all induced microshoots were cultured on MS medium at its one-quarter strength containing either IBA or NAA at 0.5, 1.5, or 3.0 mg·L~(-1). All microshoots formed roots at 0.5 or 1.5 mg·L~(-1) IBA, or 0.5 mg·L~(-1 )NAA. There were no significant differences in number of roots per shoot and length of roots among treatments. The plantlets were transplanted, acclimated in a mist system, and grown in a greenhouse. A total of 96.4% of the plants derived from tissue culture had multiple layers of ray flowers, while only 9.6% of the plants from seed propagation did. Therefore, in vitro regeneration of R. hirta ‘Plainview Farm’ was a feasible way to rapidly produce uniform plants with multiple layers of ray flowers.
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