ARS | Publication request: Protocols for Callus and Somatic Embryo Initiation for Hibiscus sabdariffa L. (Malvaceae): Influence of Explant Type, Sugar, and Plant Growth Regulators

Submitted to: Australian Journal of Crop Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 29, 2009
Publication Date: March 1, 2010
Citation: Sylvere Sie, R., Gilbert, C., Sakhanokho, H.F., Toueix, Y., Dje, Y., Sangare, A., Branchard, M. 2010. Protocols for Callus and Somatic Embryo Initiation for Hibiscus sabdariffa L. (Malvaceae): Influence of Explant Type, Sugar, and Plant Growth Regulators. Australian Journal of Crop Science. 4(2):98-105.

Interpretive Summary: Hibiscus sabdariffa, popularly known as roselle, is widely grown in tropical regions of the world. Hibiscus sabdariffa calyx has high anthocyanin content, and it is used to make tea in many regions of the world. The calyx is also used as flowering and color additive in the manufacturing of jam, liquor, and jellies. In many parts of the world, the plant is used in traditional medicine to deal with several health problems, ranging from hypertension, atherosclerosis, to cholesterol. Various pathogens adversely affect the growth of roselle plant and its seeds in various parts of the world. For example, the crop is susceptible to Fusarium oxysporum which is the main cause of diseases (foot rot and wilt) in early stages of plant development. Since all known roselle cultivars are susceptible to F. oxysporum, there appeared to be little resistance available in roselle genome. Modification of Hibiscus sabdariffa genome using genetic engineering methods would facilitate rapid development of new cultivars with traits that confer resistance to the fungus. However, in order to genetically engineer the plant, an in vitro regeneration system based for example on somatic embryo production is needed. Also, the rapid micropropagation of plants using somatic embryos can be useful for the industrial production of Hibiscus sabdariffa. Two genotypes (Hibiscus sabdariffa and Hibiscus sabdariffa var. altissima) two sugars (sucrose and glucose) and three concentrations (1 %, 2%, 3%) of each sugar, 3 plant parts (root, hypocotyl, cotyledon) were used for tissue culture. Fourteen combinations of plant growth regulators (PGRs) in MS medium and five combinations of PGR in Driver and Kuniyuki (DKW) medium were tested on hypocotyl and cotyledon for callus and somatic embryo formation. The PGR combinations used with MS medium were naphthaleneacetic acid/Kinetin (NAA/KIN), 2,4-dichlorophenoxyacetic acid/Kinetin (2,4-D/KIN), and naphthaleneacetic acid/6-Benzylaminopurine (NAA/BA) and those used with DKW medium were 2,4-dichlorophenoxyacetic acid/thidiazuron (2,4-D/TDZ). Callus formation was initiated on both genotypes with all concentrations of both sugars and PGRs and in all explant types. The best results for callus induction were achieved with 3% sucrose and the hypocotyl and cotyledon explants. Somatic embryos were obtained with DKW medium supplemented with 4 mg/l + 1 mg/l TDZ and 1 mg/l 2,4-D + 0,5 mg/l TDZ.

Technical Abstract: A significant work on callus induction and somatic embryogenesis was realized for Hibiscus sabdariffa. Two genotypes (Hibiscus sabdariffa and Hibiscus sabdariffa var. altissima) two sugars (sucrose and glucose) and three concentrations (1 %, 2%, 3%) of each sugar, 3 explant types (root, hypocotyl, cotyledon) were used for tissue culture. Fourteen combinations of plant growth regulators (PGRs) in MS medium and five combinations of PGR in Driver and Kuniyuki (DKW) medium were tested on hypocotyl and cotyledon for callus and somatic embryo formation. The PGR combinations used with MS medium were naphthaleneacetic acid/Kinetin (NAA/KIN), 2,4-dichlorophenoxyacetic acid/Kinetin (2,4-D/KIN), and naphthaleneacetic acid/6-Benzylaminopurine (NAA/BA) and those used with DKW medium were 2,4-dichlorophenoxyacetic acid/thidiazuron (2,4-D/TDZ). Callus formation was initiated on both genotypes with all concentrations of both sugars and PGRs and in all explant types. The best results for callus induction were achieved with 3% sucrose and the hypocotyl and cotyledon explants. Somatic embryos were obtained with DKW medium supplemented with 4 mg/l + 1 mg/l TDZ and 1 mg/l 2,4-D + 0,5 mg/l TDZ.