Micropropagation of damiana (Turnera diffusa), a medicinal plant
Micropropagation of the medicinally important Turnera diffusa allows exponentially faster propagation when compared to standard ex vitro propagation practices. Using the method below, up to 20 explants per parent plant can be obtained in as little as eight weeks.
Flowering Turnera diffusa.
Darklight has been working with aseptic medicinal and endangered plant species propagation for over 20 years. Moving into fungal propagation was a natural progression (or an unfortunate side-effect, you choose). Right now, Darklight is working on long-term archiving of local NNSW fungal species for future remediation and revegetation work- the culture library consists of a range of local macrofungi whose ultimate purposes have yet to be revealed.
Freshly excised nodal sections in IBA pulse media.
Turnera diffusa is a small shrub up to 2 m in size of Mexican origin. Infusions of T. diffusa leaves are traditionally employed as a relaxant and have a reputation for aphrodisiac qualities. The extracted oils are used as flavouring for many drinks.
Multiplication culture of Turnera diffusa at the three-week phase.
Pre-treatment. Non-flowering potted individuals of T. diffusa were pre-treated at 7 and 1 days prior to explant excision with a solution of 1% benomyl. During this time plants were watered at ground level only and kept indoors.
Culture sterilisation and initiation. Four node axillary branches were excised and rinsed under running water for three hours. Explants were sonicated for 1 minute to remove gross contaminants, then treated to a 30 second soak in 70% ethanol. Sterilisation was concluded by swirling explants in a 1.5% NaOCl + 0.02ml Tween 80 solution for ten minutes, then rinsed with sterile distilled water twice for five minutes each.
Explant length was reduced to three nodes in the laminar flow hood, and all were placed in MS media + 20 g/L sucrose, pH 5.8. Plants were placed under 16/8 light cycle at 25°C until aseptic culture was confirmed.
Replication. Two or three nodal lengths were placed in MS media + 30 g/L sucrose + 100 mg/L l-glutamine + 2 g/L charcoal + 1 uM NAA. Explants were placed under 16/8 light cycle at 25°C. Using a subculture period of five weeks up to 20 axillary and nodal replicants per explant could be attained. No difference in explant quality or viability is detectable between nodal or axillary explants.
Root induction. Root formation in the above replication media is spasmodic and statistically unreliable. Three node segments or axillary branches obtained from the above cultures were excised and placed in MS media + 30 g/L sucrose + 8 mg/L IBA, pH 5.8 and placed in constant darkness at 22°C. At the end of the week, all plants were transferred to a sterile mixture of damp 30% coco-coir + 30% perlite + 30% vermiculite and replaced under a 16/8 light cycle at 25°C. Once new, active growth was evident, explants were ready for deflasking.
De-flasked plantlets successfully acclimatised.
Darklight wishes to thank Torsten Wiedemann at Shaman Australis Botanicals for his help and support.
Glossary
IBA: Indole-butyric acid
MS: Murashige and Skoog Basal Medium 1962
NAA: a-Naphthaleneacetic acid
