Evaluating a DOE screen to reduce hyperhydricity in the threatened plant, Cycladenia humilis var. jonesii

Authors: PENCE, VALERIE - Cincinnati Zoo & Botanical Garden; FINKE, LINDA - Cincinnati Zoo & Botanical Garden; Niedz, Randall

Submitted to: In Vitro Cellular and Developmental Biology - Plants
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/24/2019
Publication Date: 1/2/2020
Citation: Pence, V.C., Finke, L.R., Niedz, R.P. 2020. Evaluating a DOE screen to reduce hyperhydricity in the threatened plant, Cycladenia humilis var. jonesii. In Vitro Cellular and Developmental Biology - Plants. 56:215–229. https://doi.org/10.1007/s11627-019-10038-y.
DOI: https://doi.org/10.1007/s11627-019-10038-y

Interpretive Summary: One method to save endangered plant species is propagation. Plant tissue culture is a method of propagation suitable for saving endangered plant species. Plant tissue culture is the culture of plants in sterile culture vessels such as sealed jars and tubes that contain sterile nutrient media. Large numbers of plants can be produced that are free of disease and suitable for reintroduction back into nature. However, the type of culture medium, light, and temperature conditions required to propagate an endangered plant species are often unknown and must be identified. Jones cycladenia (Cycladenia humilis var. jonesii) is an endangered plant species in the United States. Jones cycladenia can reproduce by seed but does not produce many fruits or seeds. Also, to produce seed pollination is require and its pollinators have disappeared. Further, the plant is threatened by cattle grazing and offshore vehicles. This study used advanced experimental design that simultaneously tested six factors, known to affect the growth of plants in tissue culture, to determine the factors important for propagating Jones cycladenia. Culture media and conditions were identified that reduced hyperhydricity, a major problem with previous in vitro conditions for the tissue culture propagation of Jones cycladenia. The factor that reduced hyperhydricity the most was venting the culture vessel so that there was better air-exchange.

Technical Abstract: In vitro methods for propagation and preservation are critical when seed banking is not an option for the ex situ conservation of an endangered species. Such methods rely on the ability to maintain normal growth for the ultimate goal of producing plants. Previous studies revealed that the threatened Cycladenia humilis var. jonesii can exhibit extreme hyperhydricity when cultured in vitro, but that this phenotype can be largely normalized by venting of the cultures. A design of experiments (DOE) approach was used to investigate factors contributing to hyperhydricity in this species. Using a 6-factor fractional factorial design, the effects of nitrogen, calcium chloride, 6-benzylaminopurine, gelling agent, and the presence of venting on the phenotype were tested. Venting was the strongest driver decreasing hyperhydricity, but gelling agent and other factors had significant effects, depending on the response measured. A revised medium was then designed and compared with the original maintenance medium through 2–3 subcultures. Venting was again the strongest driver decreasing hyperhydricity, but the revised medium also significantly affected some responses, even in the absence of venting. The revised medium was not optimal, as shoots became progressively smaller with each transfer, but despite their size, hyperhydricity decreased. These results demonstrate the usefulness of DOE for simultaneous testing of multiple factors, but also the need for follow-up optimization experiments. DOE can be particularly useful in working with rare species. By making efficient use of limited resources, it can facilitate the development of in vitro propagation protocols for such species and help ensure their conservation.