Callus production, suspension culture and in vitro alkaloid yields of Ephedra

Abstract

The effects of a range of plant growth regulators on callus production in various Ephedra species were examined. Species examined were E. andina, E. distachya, E. equisitina, E. fragilis var, camplyopoda, E. gerardiana, E. intermedia, E. major ssp procera, E. minima and E. saxatilis. All species produced callus on modified MS medium supplemented with 0.25 p~M kinetin and 5.0 p,M 2,4-dichlorophenoxyacetic acid or 1-naphthaleneacetic acid. Neither indole-3-acetic acid nor 3-indolebutyric acid induced significant callus formation but the latter maintained growth of established callus cultures in several species. Suspension cultures of several species were established in MS medium supplemented with 0.25 ~M kinetin and 5.0 ~M 2,4-dichlorophenoxyacetic acid or 1-naphthaleneacetic acid. Sustained fresh weight doubling times of 70 ---7 h were recorded for cell suspension cultures of E. andina grown in a semi-continuous air-lift bubble bioreactor and a minimum doubling time of 56 h was recorded for E. andina in batch culture. It also proved possible to immobilise E. andina batch cultures in sodium alginate beads. Neither parent plants or in vitro cultures of E. distachya, E. fragilis or E. saxatilis produced alkaloids. Trace quantities of 1-ephedrine and trace-0.14% dwt d-pseudoephedrine were produced by in vitro cultures of other species. The ability to produce alkaloid diminished to zero with successive subcultures.Abbreviations: Eph-1-ephedrine, Peph-d-pseudoephedrine, RGR-relative growth rate, KINkinetin, 2,4-D -2,4-dichlorophenoxyacetic acid, NAA -1-naphthaleneacetic acid, IBA -3-indolebutyric acid, IAA-indole-3-acetic acid

X Ephedra Species: In Vitro Culture, Micropropagation, and the Production of Ephedrine and Other Alkaloids

“…The former group isolated and maintained callus tissues of an unidentified Ephedra species on modified White's medium (White 1963) supplemented with 20% coconut milk and 9.uM 2,4-D. Exogenous auxin (2,4-D or NAA) was required for callus growth but the auxin IAA failed to support growth, either alone or in conjunction with a cytokinin, an observation supported in later studies (Ramawat and Arya 1976;O'Dowd et al 1993). O'Dowd et al (1993) found that IBA was similarly ineffective in the initiation of callus from stem explants of nine species of Ephedra but that it could be used to maintain established cultures when substituted for 2,4-D or NAA. It also proved to be the only auxin capable of supporting healthy, friable E. minima Hao callus growth without inducing morphogenesis.…”

“…Our studies showed that the phenylalkylamine content of plants varied greatly between species (O'Dowd 1991; O'Dowd et al 1993). Several types of variation were observed.…”

“…The first reports of the establishment of cell suspension cultures of E. foliata were by Khanna and Uddin (1976) and Uddin (1977). However, there are no descriptions of the morphology, growth kinetics or alkaloid production of Ephedra cell suspensions apart from O' Dowd (1991) and O'Dowd et al (1993).…”

“…Callus cultures from seven of our Ephedra species yielded E and/or PE but these quantities were low compared to the parent plant (Table 5) and diminished to zero with successive subcultures over 3-5 months (O'Dowd et al 1993). It is possible that alkaloid present in callus tissues in the early stages of the culture could be residual from the parent plant and that we were looking at a dilution effect over successive subcultures.…”

“…Suspension cultures have been established from a range of different Ephedra species (Khanna and Uddin 1976;Uddin 1977;O'Dowd 1991, O'Dowd et al 1993. In all cases, the medium used was MS supplemented with either an auxin (2,4-D) or an auxin (2,4-D or NAA) and cytokinin (kin).…”