In vitro propagation and leaf ultrastructure of bush tea (Athrixia phylicoides) derived from nodal explants

Abstract

Athrixia phylicoides (bush tea) is one of many plants from the Asteraceae family used as a traditional herbal medicine. With very few cultivated plants, natural growing plants currently serve as the main resource for plant material. The plant is not yet commercialised and its medicinal value is known and used only by a few people. With the long term aim at commercial scale propagation, this study consists of three parts. Firstly we developed a protocol for in vitro propagation of A. phylicoides. Secondly, the ultrastructure and morphology of leaves were studied microscopically and thirdly, comparisons were made between in vitro and ex vitro grown plants. Nodal segments of greenhouse plants were used to establish cultures. Better growth and less wilting was recorded on explants surface sterilised with NaOCl compared to Ca(OCl)2 after establishment. The addition of growth regulators IBA (indole-3-butyric acid) and BAP (6-benzylaminopurine) to the culture medium did not seem to affect the growth response of explants during the multiplication phase. Hyperhydricity was a problem throughout our trials. The development of hyperhydricity symptoms seems to be related to seasonal changes in the stock plant material used to initiate cultures, rather than the composition of growth medium or growth room temperatures. The occurrences of hyperhydricity symptoms were inconsistent and unpredictable. A rooting medium with added BAP and decreased sucrose levels resulted in a higher rooting percentage compared to the control medium, free from BAP and with a higher sucrose concentration, which yielded no rooting. However, in another experiment, in vitro rooting occurred spontaneously after subdividing and transfer of microshoots to fresh control medium. The addition of GA3 to the establishment medium (but not to the subsequently used multiplication and rooting media) yielded a slightly higher percentage of rooting. However, cultures initially established on GA3 medium yielded fewer roots per explant and roots were shorter than those of explants established on hormone free medium. The medicinal properties of plants are often linked to the production of essential oils. We hypothesised that the medicinal value of A. phylicoides can be linked to the production of the aromatic essential oils released by leaves. A microscopic study of leaves provided some preliminary insight of the mechanisms involved in the production of medicinally active products. Electron- and light microscopic examination of leaves were used to identify and study structures that are apparently involved in the production and secretion of essential oils. Two types of trichomes were identifyed – nonglandular and glandular trichomes. These glandular trichomes are multicellular with a subcuticular storage space and are present only on the adaxial surface of leaves. In the case of medicinal plants, it is essential that the medicinal properties of the plant are not altered by the method of propagation. This was our motivation for comparing the morphology and ultrastructure of leaves of plants that were grown in their natural environment to that of plants grown in vitro. Leaf surfaces of in vitro grown plants were smaller and the number of glandular trichomes per surface area was less on in vitro grown plants. There were no noticeable changes in the morphology of glandular trichomes.