Genetic stability of regenerated plants via indirect somatic embryogenesis and indirect shoot regeneration of Carum copticum L.

Ajowan (Carum copticum L.) is an important and endangered industrial medicinal plant that growing in some parts of Iran. Two efficient protocols, without somaclonal variation induction, were developed for indirect somatic embryogenesis and indirect shoot regeneration of three Iranian ecotypes of ajowan. In the first experiment, higher concentration of auxin than cytokinin (1, 1.5 and 2 mg/L of 2,4-dichlorophenoxyacetic acid along with 0.25, 0.5 and 0.75 mg/L kinetin) was used for callus induction in 5, 10 and 15 days old hypocotyl explants. In the second experiment, higher concentration of cytokinin than auxin (1 mg/L of kinetin along with 0.5 mg/L of 2,4-dichlorophenoxyacetic acid) was used for callus induction in 15-d old hypocotyl explants. The higher frequency of somatic embryos was achieved from 15 days old hypocotyl explants of Ghoom ecotype with 28.33 embryos when induced calli from MS medium supplemented with 1.5 mg/L 2,4-dichlorophenoxyacetic acid in combination with 0.5 mg/L kinetin transferred to free plant growth regulator MS medium. Momentary removing of 2,4-dichlorophenoxyacetic acid was successful for somatic embryogenesis in Iranian ecotypes of ajowan that it significantly reduce the time of the culture and thus reduce the risk of somaclonal variation. Maximum number of initiated shoots per explant was related to Shiraz ecotype with average 18.33 shoots per callus in MS medium supplemented with 1.5 mg/L of specify type of cytokinin (3-methoxy [-6-benzylamino-9-tetrahydropyran-2-yl] purine) plus 0.25 mg/L naphathalene acetic acid. The survival rate of rooted plantlets was 60.86% and 58.33% for indirect somatic embryogenesis and indirect shoot regeneration derived plants, respectively. The genetic stability of regenerated plants via indirect somatic embryogenesis and indirect shoot regeneration was proved through flow cytometry analysis.