Eremophila Species (Poverty Bush; Emu Bush)

Distribution and Importance of Eremophila Species

The genus Eremophila (Myoporaceae) consists of woody shrubs and trees which typically grow in low rainfall areas and are characterized by the viscid to resinous vegetative parts, ebracteate flowers and indehiscent woody fruit. In terms of biogeographical distribution, Eremophila is one of the most significant Australian desert genera. Of the 210 Eremophila species recognized by Chinnock, 175 occur throughout Western Australia. Seventy-five percent of the species are entomophilous, the remainder being ornithophilous. The genus is an important component of the semi-arid vegetation of pastoral zones and many species are browsed by animals when the plants are at the seedling stage. Some Eremophila species, e.g. E. gilesii F. Muell. and E. mitchellii Benth., are regarded as woody weeds. Many species occur on impoverished soil and, as a consequence, they are collectively referred to as poverty bush. Since emus favour the fruits of some Eremophila species, the term emu bush is also commonly used.

Eremophila species have been highly valued for medicinal and cultural purposes by the Aboriginal people in central Australia. The use of different species in the cure or alleviation of a number of disorders, rheumatism, colds, influenza, fever, scabies, insomnia, and internal pain has been documented. They have also played a role in the ceremonial life of the Aboriginal people. For example, E. longifolia (R. Br.) F. Muell. is considered to be the most sacred and mystical of all central Australian plants. Many Eremophila species are currently recognized for their horticultural and floricultural potential due to their showy and spectacularly coloured flowers and calyx. In recent years, a number of species, particularly E. glabra (R. Br.) Ostenf, E. maculata (Ker Gawler) F. Muell. and E. racemosa (Endl.) F. Muell., have been grown as ornamentals in America and Europe. In Australia, 61 species are currently in, or potentially of, horticultural use. They are a useful shrub and small tree in revegetation programs in rangeland and minesite environments because of their tolerance to drought, fire, frost and grazing. Several Eremophila species are now considered rare and endangered as a result of industrialization and farming practices.

Propagation of Eremophila and Production of Secondary Metabolites

Plant establishment of Eremophila species from fruits is difficult. Although propagation can be achieved from seeds, cutting or grafting, seed germination is extremely variable due to the woody nature of the fruit, the seeds being released when the fruit wall decays. The fruits are dry, drupe-like with an exocarp and endocarp which appear to play a controlling role in seed emergence. Depending on the species, the fruit can contain from 2 to 16 seeds. Germination is restricted primarily by physical factors, although evidence for a secondary chemical inhibitor is increasing. The main propagation technique for establishing Eremophila is from cuttings. Where this is not successful, grafting may be attempted. Propagation by tissue culture has been tested and seems to offer an alternative. In an early experiment, Dell (1978) established stem calli of E. fraseri F. Muell. Young, green stems, after sterilization (1% aqueous NaOCl for 20 min) and rinsing in 70% ethanol and sterile water, were cut into 0.5-1 cm length, partitioned lengthwise, and inserted into either 7.5-ml agar slopes or plastic Petri dishes containing SH medium. Cultures were kept at 25 °C under continuous fluorescent light (0.02-0.03 Einsteins). Although callus was formed without the addition of growth hormones, inclusion of 2,4-D and kinetin in the medium greatly enhanced growth rates with maximum observed for 0.1 mg/1 of 2,4-D and kinetin. Callus tissue was initially formed from proliferation of cambial and cortical cells. The calli were initially white but turned green, due to the formation of chlorophyll, even throughout periods of maximum growth. During growth experiments, leaves and shoots appeared from the surface of calli with both nodal and smooth surfaces but shoot formation was inhibited by high concentrations of 2,4-D. Within 6 weeks of subculturing, stem primordia arose endogenously in callus. Recent studies have indicated that it is possible to propagate E. ‘mirabilis’ (Chinnock MS) and E. nivea Chinnock by tissue culture and this method is being applied to the propagation of some rare and endangered species, e.g. E. viscida Endl.. For example, the in vitro generation of seedlings and propagation of E. ‘mirabilis’ by tissue culture is described (Fig. 2) (G. Richmond pers. comm. 1993). Eremophila fruits are characteristically indehiscent, dry and drupe-like. The fruits of E. ‘mirabilis’ are ovate, grey-brown in colour, 5-8 mm in length and 3-5 mm in width. Each fruit possesses four locules, each containing two to three seeds. The seeds are narrowly obovate, orange-white, 2.5-3 mm in length and 1 mm in width. Working in a laminar flow cabinet, the fruits were cut open, the seeds excised using tweezers, surgical scalpel, dissecting needle and a razor blade, and, after examination of each seed under a dissection microscope, healthy, firm, plump seeds were selected. These were surface sterilized with 10% aqueous sodium chloride and cultured on Murashige-Skoog (1962) minimum organics medium (MSMO) modified by supplementing with kinetin (0.2 mg/1) and sucrose (2% w/v) and adjusted to pH 5.8 with 1 M potassium hydroxide. Nodal tissues from seedlings of E. <mirabilis> grown in vitro, were cut into segments (1-2 mm), explanted on Murashige and Skoog medium containing kinetin (0.2 mg/1) and incubated at 28 °C with 2500 lx illumination and 16-h photoperiod. Axillary shoots developed strongly on this medium. After 3-4 weeks, the axillary shoots were excised and rooted on an MSMO medium supplemented with indolebutyric acid (1 mg/1) and sucrose (2% w/v) at pH 5.8 Rooted shoots were removed from flasks and hardened off in a glasshouse. The suckering habit of E. ‘mirabilis’ was evident from the adventitious in vitro regeneration of suckers from lateral roots. Similar propagation of the rare and endangered species E. nivea and E. viscida appears promising. Chemical interest in Eremophila began in 1910 when E. maculata, considered to be poisonous to stock, was found to produce a cyanogenetic glycoside which was later shown to be prunasin. It was further stimulated in the 1930s with the isolation from Eremophila mitchellii of the sesquiterpene eremophilone, at that time the only structurally atypical naturally occurring sesquiterpene known. Eremophilone was considered to be a substitute for sandalwood oil. Since then a large number of desert-adapted Eremophila species have been shown to produce high yields of resins which are mostly composed of flavones and terpenoids. Many of these terpenes are unique and reflect unusual biosynthetic pathways. The thickness, distribution and the amount of resin vary considerably, 5-10% being typical, although in a number of cases the resin constitutes up to 20% of the dry biomass. Trichomes on the leaves are considered to be responsible for the production of the resin. Since many compounds produced by trichomes are biologically active, providing ‘a first line of defence for the plant’, this suggests that such compounds may exhibit antiviral, antibacterial, antifungal and insecticidal activities. Eremophila species can be considered good sources of flavones, some sesquiterpenes and a variety of oxygenated diterpenes. Members of the genus Eremophila are thus important arid-zone species offering potential in the areas of horticulture, rangeland management, soil conservation and as a source of renewable raw materials.

Concluding Remarks

Tissue cultures of Eremophila species were established initially with the objective of studying the biosynthesis of the unique and structurally interesting terpene metabolites produced by the whole plants. Although this aim was not realized, the observation that callus cultures of several species produce significant amounts of verbascoside was as interesting as it was unexpected. The highest yield of verbascoside expressed (20.8% dry wt.) was from callus cultures of E. decipiens established and maintained under the following conditions. Leaves were washed under running water for 20 min, sterilized (1% NaOCl solution) for 20 min and rinsed (x3) in sterile double deionized water. Explants were trimmed to 10 x 5 mm segments and cultured in MS medium modified in the following way: reduction of Mg2+ from 1.50 mM to zero, addition of thiamine HC1 (5 μM), inositol (550  μM), nicotinic acid (30  μM), pyridoxine HC1 (8  μM) and sucrose (30 g/1) and adjusted to pH 6.1. Maximum growth of callus was achieved with 1 mg/1 of 2,4-D and no kinetin. The cultures were maintained at 25°C under continuous fluorescent lighting (Growlux). Callus initiation took 1-3 weeks and the first subculture was carried out at 6 weeks. For the isolation of verbascoside, callus tissue (10-100 g wet weight) was homogenized in a solution of dichloromethane : methanol (1:1). The methanol layer was evaporated and the residue re-extracted with ethyl acetate. Analysis of the ethyl acetate-soluble fraction by TLC and NMR indicated that this fraction was mostly verbascoside.

If, as it appears, verbascoside proves to have a pharmacological application, then Eremophila cultures become a useful addition to that select group of species which are high producers of verbascoside. In addition, given the relative difficulty of classical means of propagation of Eremophila species, tissue culture techniques may provide a more reliable method. This would have significant value in the deployment of the hardier of these species in rangeland management and land reclamation. Also important is the possibility of utilizing the resin-producing xerophytic species as sources of renewable chemicals that could be used for feedstocks for speciality chemicals.