Native four o’clocks (Mirabilis spp.) are drought-tolerant perennials native to North America, valued for their vibrant flowers and ecological contributions, including pollinator support and soil stabilization. Despite their ornamental appeal and value in ecological restoration, their use in horticulture has been limited due to propagation challenges such as low seed availability, poor germination, and complex dormancy requirements. Micropropagation, the production of new plants from small tissue sections under sterile conditions using artificial media and controlled environments, presents a promising solution to these limitations. It offers a reliable method to produce large quantities of native Mirabilis for conservation and nursery applications. In 2021, an effective micropropagation protocol was developed for the rare North American species Mirabilis macfarlanei. Building on this success, the current project evaluated the applicability of this protocol to three additional native species: Mirabilis greenei, Mirabilis laevis var. villosa, and Mirabilis laevis var. crassifolia. Single-node cuttings were surface sterilized with 20% bleach and cultured on Driver and Kuniyuki Walnut (DKW) medium supplemented with 4 µM 6-benzylaminopurine (BA). All three species were successfully established in vitro and proliferated. Growth parameters, including average number of shoots, shoot length, and shoot dry weight, were compared between DKW with 4 µM BA and Murashige and Skoog (MS) medium with the same BA concentration. For M. greenei, shoot proliferation was similar between MS and DKW (5.7 and 5.8 shoots, respectively). M. laevis var. villosa and M. laevis var. crassifolia produced more shoots on MS medium (10.6 and 4.5) than on DKW (7.8 and 3.9). All species exhibited higher shoot dry weights on MS medium, while shoot lengths were comparable across treatments. Rooting success varied among species. The rooting medium developed for M. macfarlanei (DKW supplemented with 0.5 mg/L indole-3-acetic acid [IAA], 0.5 mg/L naphthaleneacetic acid [NAA], and 0.15 mg/L Sequestrene® 138) was effective only for M. greenei. However, M. laevis var. villosa and M. laevis var. crassifolia rooted better on MS medium containing 1 µM NAA and 0.15 mg/L Sequestrene® 138. Rooted plantlets of all three species were successfully acclimatized in rockwool, with M. laevis var. villosa and M. laevis var. crassifolia flowering under greenhouse conditions.
