Domestication and crop development of Duboisia spp. (Solanaceae)
Tropane alkaloids have always played a significant role in ethnomedicine as well as orthodox medicine. Formerly, the main sources were the genera Datura, Brugmansia, and Atropa of the Solanaceae family, but recently the Australian genus Duboisia, especially the species D. myoporoides and D. leichhardtii, have become an important source. D. myoporoides is a small tree of the rainforest areas of the eastern coast of Australia; D. leichhardtii prefers a slightly drier habitat further inland. Dried leaves are used for the extraction of the pharmaceutical products.
It was during World War II that Duboisia leaf was collected in quantity and this new source of pharmaceutical products gained in importance. Research activities decreased after 1945 and ceased about 1954, leaving a few plantation trials involving several hybrids between D. myoporoides and D. leichhardtii. In the following years, what little effort continued in further crop development came mainly from individual growers.
In the mid 1960s Boehringer Ingelheim, a German pharmaceutical company and the main buyer of Duboisia leaf, decided to resume the work done in Australia during the war. Available hybrids, as well as new accessions from the natural populations, were screened and a breeding programme established. The selection criteria used were alkaloid pattern and content, leaf yield, shoot regrowth from stumps, drought resistance, insect resistance, wind resistance, and suitability for mechanical harvest. Simultaneously, research was initiated to develop techniques for vegetative propagation and for weed and pest control, while improvements in cropping and fertilizer use were achieved through experimentation. In addition, machinery for planting, maintenance and the harvesting was adapted or designed.
Since 1980, raw material of Duboisia for export has been derived from cultivated plants only, and production has proven to be profitable.
The family Solanaceae provides numerous medicinal plants. A prominent group is the tropane alkaloids, containing the main genera-Datura, Brugmansia, Atropa, Hyoscyamus. These have played significant roles in ethnomedicine and religious practices of the Old and New World and are now also important in orthodox medicine.
The main tropane alkaloids used in orthodox medicine are atropine (dl-hyoscyamine) and scopolamine (hyoscine). Atropine as a parasympatholytic dilates the pupils of the eyes; it also serves as an antidote against toxins such as phosphoric acid esters like E 605. Formerly scopolamine was used mainly in association with morphine as an analgesic at childbirth and as a hypnotic agent in certain mental disorders. During World War II, scopolamine was found to be of great value in the treatment of bomb shock and against sea- and air- sickness. Demand increased, and since access to the traditional sources was restricted, new sources were sought, leading to interest in the exploitation of Duboisia species.
The genus Duboisia comprises three species: D. myoporoides R. Br., D. hopwoodii F. Muell. and D. leichhardtii F. Muell., discovered in 1802, 1861 and 1867, respectively. The occurrence of these woody representatives of the Solanaceae family is restricted to Australia and New Caledonia (D. myoporoides only). D. myoporoides occurs along the east coast of Australia in frost-free areas with an annual rainfall exceeding 550 mm. The occurence of D. leichhardtii is restricted to a relatively small, drier inland region in South-East Queensland. Both typically are found as isolated trees at the edge of the forest. Due to their corky bark they easily withstand bush fire, and when felled they sucker profusely, which enables them to regenerate quickly in disturbed areas (Barnard,1952). In about 1880, scopolamine and hyoscyamine were identified as the main active principles of pharmaceutical importance in the leaves of D. myoporoides and D. leichhardti. In D. hopwoodii, nicotine and related compounds were found prevalent.
D. myoporoides and D. leichhardtii are trees that grow up to about 14 m tall. The wood is extremely light and the stem bark very corky (hence its local name `corkwood'). The leaves are glabrous, broad-lanceolate to obovate in D. myoporoides, while in D. leichhardtii they are narrower, more lanceolate (6-10 cm long) and light green. Due to its narrower leaf, D. leichhardtii appears less leafy. Both species flower profusely in spring, with small, white, typically solanaceous flowers in cymose panicles. The fruit is a small globular berry about 5 mm in diameter, containing up to 15 or more seeds.
In 1940 collection of Duboisia from the wild commenced. Commercial production of scopolamine began in 1941, that of hyoscyamine /atropine in 1942 (Barnard 1952). Propagation of plants and plantations was limited in the early years due to the abundance of naturally occurring trees when land was cleared and burnt. For the harvest of Duboisia leaf for extraction, the trees are cut, the complete branches with the leaves are air dried and then the dry leaves removed (traditional method), or the fresh branches are chopped, the leaf separated from the wood chips and dried artificially (common industrial method). The stumps normally regrow and can be harvested again after about 12 months. The first large- scale plantations did not occur until the late 1950s, and then they were mainly from seedlings transplanted from natural germination in the wild.
Plant-related research initially focused on plant propagation, on identification of alkaloid-rich genotypes, and the elucidation of the reasons of alkaloid variation (Barnard and Finnemore 1945). These activities gained a new dimension when, in the area where the habitats of D. myoporoides and D. leichhardtii overlap, intermediate types were found that appeared to be natural interspecific hybrids. Artificial hybridization experiments started, and by 1945 the first hybrid plants were established in the field (Groszman et al. 1949; Hills et al. 1954a, b, c). After the end of World War II, commercial alkaloid extraction and government-supported research continued until about 1954 when the export embargo on Duboisia leaf was lifted. Commercial extraction was no longer competitive in the international market; research was considerably reduced if not ceased. Exportation of Duboisia leaf from collection of wild material and increasingly from cultivation commenced and continued with a steadily growing level, one of the main buyers being Boehringer Ingelheim.
Domestication and crop development of Duboisia spp. did not lead to an agroforestry production system. However, this minor forest species turned into a commercial plantation crop in a relatively short time and may well serve as an example for the problems and opportunities to be expected in similar projects. In other new medicinal crops, agroforestry may be the appropriate production system.
In 1951, Boehringer Ingelheim, producer of pharmaceuticals and fine chemicals with a long tradition in alkaloid extraction, launched Buscopan®, a derivative of scopolamine. This compound is a potent spasmolytic and analgesic, lacking the psychotropic effects of scopolamine. Demand for raw material increased considerably, and to secure the supply of high-quality raw material the company decided to develop its own plantations. In the late sixties its R&D work on Duboisia started.
The plant population present in the existing plantations showed considerable variation. In addition to seed-propagated stands, clones were present derived from early hybridization work and from superior natural sports. After establishing appropriate facilities for analysis, some 20 future parent plants were selected from this highly variable population, together with a small number of pure D. myoporoides and D. leichhardtii specimens.
Insufficient germination and extremely low striking rates of cuttings were common problems encountered by the Duboisia growers. Consequently, seed germination trials were established, which confirmed the existence of a seed dormancy period and led to seed pretreatment methods, which resulted in a germination rate of 80-90%. Similarly, after a series of vegetative propagation trials, methods were optimized with respect to plant parts used, season, cutting techniques, disinfection, applications of rooting hormones, substrate, potting material, greenhouse climate and hardening off procedures. Standard striking rates of 90- 99% were achieved. More recently, a method for tissue culture propagation has been established and implemented.
Only conventional breeding methods were applied. Special techniques of controlled crossing of Duboisia have been developed, the most important point being the synchronization of flowering. In the course of a continued crossing programme, genotypes were identified which carried useful and highly heritable traits, thus building up a pool of parent plants. Out of more than 12 000 progeny from controlled crosses, about 20 individuals have been cloned for commercial cultivation.
Selection criteria (in the order of the stages of selection) were alkaloid content, alkaloid pattern, ease of vegetative propagation, regrowth potential, leaf yield, stability of alkaloid pattern, drought resistance, wind resistance, frost resistance, insect and nematode resistance, and suitability for mechanical harvest.
Unexpected changes were frequently found in alkaloid content and pattern. For example, strong genotype x site interaction and declining alkaloid content of ageing trees were observed. These and similar phenomena made clonal selection, as well as yield estimates, unpredictable. Therefore, the patterns of various alkaloids (scopolamine, hyoscyamine and some other accompanying minor alkaloids) from Duboisia hybrid individuals and clones were assessed with respect to-
· variation between genotypes
· variation within hybrid progenies of crosses
· intraclonal variation
· variation over several generations of clonal propagation
· variation over productive life cycle (up to 10 years)
· seasonal variation
· variation between locations
· effects of fertilizer, soil types, and water supply
As a result, clones were identified in which the genetic traits of alkaloid content were stronger than usual, so that the impact of environmental and other factors was reduced and alkaloid yield became more predictable. Harvesting has been closely adapted to seasonal variations in alkaloid content.
Duboisia cultivation practices used to be rather extensive with wide spacing, little or no use of fertilizer and utilization of suboptimal soil types. These factors kept yields low. With the aim of more intensive land use, crop requirements were assessed in field trials with respect to soil type, soil nutrients, fertilizer application, cover crops and planting density. High importance was given to weed control, this being a major cost factor. In cooperation with the regional distributors, herbicides were tested for use in Duboisia and eventually registered. Setbacks, such as accumulation of subcritical amounts of residues over a long time, led to the identification of a well-balanced weed control system, which combined mechanical weeding with an essentially minimum level of herbicide application.
As has to be expected when establishing pure stands of plants normally growing in biologically diverse natural stands, insects and parasites feeding on them became pests. However, appropriate crop rotations and the selection of tolerant clones has resulted in the reduction of these problems. For example, nematode populations have been reduced to an acceptable threshold level by this method. Similarly, the incidence of a weevil `stem borer', whose larvae mine the trunk, has been greatly reduced after clones with a different type of bark were introduced, while another damaging insect, the `flea beetle' Psylliodes parilis, is currently under investigation and cultural practices to reduce its impact are in sight.
Harvesting of Duboisia used to extend over the whole summer period, but through studies of the seasonality of alkaloid content, it became possible to define clone-specific periods of maximum alkaloid content. Special high-capacity harvesting machines have been developed, that eliminate the need for protracted manual harvesting and allow the harvest to take place when alkaloid contents are high.
Leaf-drying capacity must match harvesting capacity. Dryer systems were tested and developed for increased drying quality and throughput, and for the avoidance of dust from dried plant material. This has helped to minimize the exposure of workers to the pharmaceutically active substances.
A number of Duboisia hybrid clones have been developed that are superior to the common `standard' hybrid in scopolamine content (about double), improved growth characteristics, predictability of quality and yields, and lower susceptability to soil nematodes and stem borers.
Cultural practices have been improved with respect to labour requirements, use of agrochemicals and soil conservation. There is a better understanding of insect population dynamics, leading to a more rational use of pesticides. Health hazards originating from the plant material have been greatly reduced.
Today Duboisia is a commercially viable crop, which contributes considerably to exports from the region, and is the main source of raw material for tropane alkaloid production worldwide.
Several factors contributed to the progress achieved:
· There was basic knowledge of the plants and their cultivation available from farmers and research institutions that provided the starting point for Boehringer Ingelheim´s domestication efforts.
· Rapid progress was made in quantitative analysis of alkaloids and its use in a high-capacity analytical laboratory. This was a precondition for an efficient screening of plant material.
· The combination of commercial farming and R&D activities provided the indispensable immediate feedback and long-term continuity that are necessary when dealing with plantation crops. From identification of suitable parent plants to the first commercial harvest of a new clone takes at least 8-10 years.
· .Considerable support, especially in phytopathology was granted by the Queensland Department of Primary Industries. Research on insect biology was carried out at the University of Queensland.
Barnard C. 1952. The duboisias of Australia. Economic Botany (1): 3-17.
Barnard C. & Finnemore H. 1945. Drug plant investigation: 1. Progress report. Journal of the Council for Scientific and Industrial Research (Australia) 18(4):277-285.
Groszmann H.M., Kelenyi G.P. & Rodwell, C.N. 1949. Hybrids between Duboisia mycoporoides and D. leichhardtii. Queensland Journal of Agricultural Science 6(3):1-8.
Hills K.L., Bottomley W. & Mortimer P.I. 1954a. Variation in the main alkaloids of Duboisia myoporoides R.Br. and D. leichhardtii F.Muell.: 2. Duboisia myoporoides. Australian Journal of Applied Science 5(3):258-275.
Hills K.L., Bottomley W. & Mortimer P.I. 1954b. Variation in the main alkaloids of Duboisia myoporoides R.Br. and D. leichhardtii F.Muell.: 3. Duboisia leichhardtii. Australian Journal of Applied Science 5(3):276-282.
Hills K.L., Bottomley W. & Mortimer P.I. 1954c. Variation in the main alkaloids of Duboisia myoporoides R.Br. and D. leichhardtii F.Muell.: 4. Interspecific hybrids. Australian Journal of Applied Science 5(3):283-291.