Micromorphology and phytochemistry of the foliar secretory structures of Stachys natalensis Hochst. and development of an in vitro propagation protocol.

i Many members of the genus Stachys have been used as ornamental plants, or as edible foods and, in a number of cultures, as traditional medicine. One such species, Stachys natalensis Hochst. , is a perennial, straggling shrub with aromatic leaves that are covered with hairs. Despite its widespread use for a number of reasons, little information has been available on this plant thus far, its foliar secretory apparatus, or the nature and potential therapeutic value of these secretions. Hence, the aims and objectives of the present study were to elucidate key micromorphological features of the leaf secretory structures of S. natalensis, using both light and electron microscopy and to determine the location and chemical composition of the exudates using various histochemical and phytochemical techniques. An additional aim was to establish an in vitro micropropagation protocol for the sustained and high-yielding production of this elusive and often difficult to cultivate species. Furthermore, the foliar micromorphological fidelity between field and in vitro propagated material was compared. Electron micrographs indicated the presence of glandular and non-glandular trichomes on both abaxial and adaxial foliar surfaces of field grown plants. Greater trichome density was observed on the abaxial surface with trichome distribution decreasing as the leaf developed (p<0.05). Uniseriate, unbranched and striated non-glandular trichomes, present on the foliar surfaces of S. natalensis are known to play a role in physical plant defence mechanisms against herbivory. The presence of an elevated cellular pedestal and striated micro-ornamentation on the stalk served as characteristic features of the non-glandular trichomes. Peltate and capitate glandular trichomes were also identified on the foliar surfaces across all developmental stages. Peltate glands consisted of a short stalk and a multicellular head containing two to eight secretory cells. Two types of capitate trichomes were observed. Type I capitate trichomes consisted of a striated stalk, cutinized neck cell and bulbous head which may be unior bi-cellular;Type II capitate trichomes were comprised of a wide base, long and tapering, septate stalk, neck cell and a multicellular secretory head cell. The process of secretion differed between the peltate and capitate glandular trichomes. The peltate and Type II capitate trichomes included a porose cuticle which facilitated the release of secretion to the exterior. Cuticular rupture at weak points of the equatorial plane of the secretory head was observed in Type I capitate trichomes. Qualitative histochemical staining of leaf sections and preliminary phytochemical tests revealed the presence of alkaloids, lipid components, terpenoids and complex polysaccharides concentrated in the glandular trichome head cells and leaf crude extracts, respectively. The perceived therapeutic benefits of this plant are likely to lie within this suite of secondary metabolites. Stachys natalensis plant extracts also contained considerable levels of total phenolic compounds (3.43 ± 0.01 mg GAE/g dry material) and flavonoids (3.04 ± 0.01 mg QE/g dry material). The methanolic extracts demonstrated significant free radical scavenging ability (49.49 ± 3.87 ug/ml) which indicates the potential for its use as a natural antioxidant. ii In vitro propagation protocol using axillary bud explants was developed for this species. A multi-step decontamination treatment involving explant immersion in 1% and 3% NaClO, followed by 0.1% HgCl2 was the most efficient method for explant decontamination, resulting in overall explant survival of 48%. All media preparations resulted in > 70% bud break within three weeks with cultures initiated on Medium C ( MS supplemented with 0.5 mg/l BAP and 0.5 mg/l IBA) showing the highest percentage of bud break. Growth medium B (0.5 mg/l kinetin and 0.5 mg/l IAA) showed the greatest total shoot multiplication, number of shoots/explant (9.1 ± 3.6) and height/explant (50.2 ± 5.0 mm) compared to other PGR combinations after 12 weeks. The addition of exogenous auxin (2 mg/l IAA) to MS medium allowed for 64% of plantlets to produce adventitious roots in five weeks, after which rooted plants were acclimatized. Acclimatized plantlets (92 ± 4.2 %) did not show any gross morphological abnormalities compared to field-grown plants, apart from the presence of visibly longer non-glandular trichomes. The peltate and both subtypes of capitate glandular trichomes of acclimatized plants were morphologically similar to their field-grown counterparts. Trichome density on acclimatized plants was greater on the abaxial surface of emergent leaves and this density decreased with leaf maturity, as was observed with field-grown plants. This study appears to be the first investigation of the micromorphology of the foliar structures of S. natalensis. Future studies on morphological aspects of secretory structures should include cytochemical investigations to determine the exact mechanism and origin of glandular secretions. Further analyses regarding the composition of the glandular essential oils and its potential pharmacological efficacy are required. With an effective in vitro propagation protocol being presently established, further optimisation with respect to the type and concentration of exogenous PGRs, explant type or even various routes of organogenesis can be investigated. This may provide a means of enhancing plantlet production, maintaining superiorselected genotypes, and thus potentially maximising the yield of putative pharmacologicallyimportant secondary metabolites.