Establishment of an In Vitro Micropropagation Protocol for Hibiscus moscheutos L. ‘Berry Awesome’

Abstract

Hibiscus moscheutos L. ‘Berry Awesome’ is a complex hybrid of the new Proven Winners Summerific series of varieties with highly ornamental characteristics. Micropropagation of highly ornamental varieties is important for mass production of planting material for commercial purposes. The traditional methods for propagating Hibiscus varieties, such as cuttings or seed propagation, however, do not guarantee high rates of production of high-quality seedlings. To solve this problem, an attempt was made to develop protocols for micropropagation of Hibiscus moscheutos L. ‘Berry Awesome’ in vitro on agar and liquid medium using a bioreactor system, followed by ex vitro adaptation of the regenerants. The optimal method for sterilization of nodal explants as well as the optimal composition of the initiation medium for shoot proliferation and rooting were determined. For micropropagation on a liquid medium, a rocker-type bioreactor was used, and its advantages over micropropagation on an agar medium were demonstrated. The results showed that the best sterilization method for nodal segment explants was as follows: pretreatment by rinsing with running tap water, sterile water, and distilled water for 70 min and soaking for 5 min in a mixture of solutions of ethyl alcohol (96%), hydrogen peroxide (38%), and water in a ratio of 1:1:2. In this case, live and sterile explants accounted for 62.6%. The optimal initiation medium for axillary buds in nodal segments was the Murashige and Skoog (MS) medium supplemented with 0.1 mg L−1 N-(2-chloro-4-pyridinyl)-N’-phenylurea (CPPU), which resulted in 73.3% of axillary buds being induced. The optimal solid proliferation medium was MS medium supplemented with 0.1 mg L−1 CPPU with a proliferation coefficient of 5.8. In a liquid medium, the optimal concentration of CPPU was 0.05 mg L−1 with a proliferation coefficient of 9.2. The best medium for rooting/shoots with agar and in bioreactors was MS medium with the addition of 0.1 mg L−1 indole-3-butyric acid (IBA). The highest rooting rate was 99.0% in both types of media, and the survival rate of plantlets was 88.7% in solid media and 98.7% in the bioreactor.