The presence of a raised cellular base and striped micro-ornaments on the stem were characteristic features of the non-glandular trichomes. Cuticular breakage at weak points of the equatorial plane of the secretory head was observed in Type I capitate trichomes.
39 Figure 4.6 Average density of non-glandular (A) and glandular trichomes (B) in three stages of leaf development (emergent, young and mature) and on abaxial and adaxial leaf surfaces of S. 70 Figure 4.22 Average non-glandular [ A ] and glandular trichome density [B] at three stages of leaf development (emergent, young and mature) and on abaxial and adaxial leaf surfaces of acclimated S .
INTRODUCTION
- TRADITIONAL MEDICINE IN SOUTH AFRICA
- OVERVIEW OF THE GENUS STACHYS
- DESCRIPTION OF STACHYS NATALENSIS
- RESEARCH RATIONALE AND MOTIVATION
- AIMS OF THE PRESENT STUDY
- OBJECTIVES
Legkobit and Khadeeva, 2004; Ghiorghita et al., 2011), but without reference to the reproduction of S. . natalensis seedlings en masse. This study was undertaken to describe key micromorphological features of S. natalensis leaf structures, elucidate the composition and localization of leaf exudates, and create a reliable in vitro micropropagation protocol for S. natalensis.
LITERATURE REVIEW
- TRICHOME MORPHOLOGY
- Non-glandular trichomes
- Glandular trichomes
- TRICHOME FUNCTIONS
- Plant defence against herbivory
- Pollination
- Protection against water loss, UV-B and light damage
- STACHYS: HISTORY AND BIOLOGICAL ACTIVITY
- BIODIVERSITY IN SOUTH AFRICA
- PLANT TISSUE CULTURE
- PROCESS OF IN VITRO MICROPROPAGATION
- Stage 0: Selection and maintenance of stock plant
- Stage I: Initiation and establishment of an aseptic (sterile) culture
- Stage II: Shoot multiplication
- Stage III: Rooting
- Stage IV: Establishment of plantlets in soil
10 Vernonia amygdalina (Adeyemi, 2011) and Aphis nerii, an arthropod fatally immobilized in the sticky exudate of the gland trichomes of Sicana odorifera (Cucurbitaceae) (Kellog et al., 2002). Cytokinins are involved in the stimulation of cell division and direct morphogenic responses (George et al., 2008).
MATERIALS AND METHODS
- SAMPLE COLLECTION AND PREPARATION
- STEREOMICROSCOPY
- SCANNING ELECTRON MICROSCOPY (SEM)
- Freeze-drying
- Chemical fixation
- Cryo-SEM
- LIGHT MICROSCOPY
- Wax embedding
- De-waxing protocol
- HISTOCHEMISTRY
- PHYTOCHEMICAL PROFILE
- Solvent extraction
- Qualitative phytochemical tests
- Quantification of total phenolics and flavonoids
- Antioxidant activity
- MICROPROPAGATION OF STACHYS NATALENSIS
- Plant collection and decontamination
- Culture conditions and media
- STATISTICAL ANALYSES
Dragendroff's test: Two drops of Dragendroff's reagent were added to one ml of the extract. Ferric trichloride test: Two ml of distilled water were added to 1 ml of the extract and a few drops of 10% ferric trichloride.
RESULTS
TRICHOME TYPES AND DISTRIBUTION
B] The abaxial surface of a mature leaf shows non-glandular trichomes, often occurring on the central midvein and branch veins. D] Mature leaf showing glandular (black arrow) and non-glandular (white arrow) trichomes on the abaxial surface. 36 Figure 4.2 SEM micrograph showing an overview of different types of non-glandular and glandular trichomes on the abaxial surface of an emerging leaf, including: elongated non-glandular trichomes (NG) and three conspicuous types of glandular trichomes viz.
TRICHOME MORPHOLOGY
A] Cryo-SEM image of peltate trichome in epidermal depression with single stalked cell (SC) found on the abaxial surface of a mature leaf. B] Peltate trichomes with multicellular (four-celled) head found on the abaxial surface of a young leaf. C] Multicellular (eight or more cells) head of peltate trichomes on the abaxial surface of an emerging leaf specimen.
A] Type I glandular capitate trichome with tuberous head cell, distinct neck cell (NC) and shorter stalk without septum on abaxial leaf surface. B] Type II glandular capitate trichome on the adaxial surface, characterized by a disc-shaped multicellular head (MTH) and an elongate septate stalk (SS) that appears bulbous at the base and tapers toward the apex. A] Peltate trichome in the presecretory phase (PSP) showing accumulation of secretion in the subcuticular space in the glandular head cell.
C] Type II main trichome in the process of secretion via openings (SO) on the surface of each of the multicellular head cells. D] Cellular rupture (CR) occurring at fragile regions in the head cell of Type I capitate trichome.
HISTOCHEMISTRY AND PHYTOCHEMISTRY
B] Ruthenium red stained positive for mucilage and pectin in multicellular head of peltate trichomes (pink-red). E] Positive Nadi reaction in head cells of peltate trichome (red) for the presence of oleoresins.
MICROPROPAGATION OF STACHYS. NATALENSIS
- Decontamination and culture establishment
- Shoot multiplication
- Rooting and acclimatization
Mean values sharing the same letter within a column are not significantly different (p<0.05) according to Tukey's post-hoc test. Growth medium B (kinetin and IAA) showed the greatest total shoot proliferation and elongation (after the bud break stage) compared to other PGR combinations after 12 weeks (Figures 4.15 and 4.16). Average shoot production per planting and shoot height per planting were assessed using a one-way ANOVA (Table 4.5).
Plants showed significant shoot multiplication and elongation in medium B compared to other growth media and were transferred to medium containing only PGR IAA (2 mg/l) to promote adventitious root formation. Plants with a well-developed root system and root length > 30 mm were selected for acclimatization (Figure 4.17). A] Medium A supplemented with 2 mg/l BAP and 1 mg/l IAA showing relatively slow shoot multiplication (Bar: 10 mm).
B] Medium B supplemented with 2 mg/l kinetin and 1 mg/l IAA showing significant proliferation and elongation of healthy microshoots (Bar: 10 mm). 63 Figure 4.16 The effect of different PGR combinations used in the propagation stage on the average number of total shoots produced from all surviving explants over a period of 12 weeks.
MICROMORPHOLOGY OF SECRETORY STRUCTURES OF IN VITRO PROPAGATED
- Non-glandular trichomes
- Glandular trichomes
- Trichome distribution and density
66 Figure 4.19 SEM micrograph of elongated non-glandular trichome on abaxial surface of freeze-dried emergent leaf of acclimated S. Emergent leaves of acclimated plants appeared to have a dense cover of trichomes which decreased markedly with leaf maturation (Figure 4). Trichome densities between three growth stages (emergent, young and mature) and two leaf surfaces (abaxial and adaxial) of acclimated plants were investigated using a two-way ANOVA (Figure 4.22).
Multiple comparisons of the three developmental stages using Tukey's post-hoc test indicated that these differences in trichome density existed between emerging and young leaves, emerging and mature leaves and young and mature leaves (Glandular and non-glandular: p<0.05). The abaxial surface was found to contain a greater number of glandular trichomes, while the adaxial surface had a slightly greater non-glandular trichome density (Table 4.6). Trichome density between wild-grown and acclimated plants at different developmental stages (emergence, young, mature) was determined using a two-way ANOVA.
In both cases, trichome density was significantly higher on emerging leaves, and decreased as the leaf matured (Figure 4.23). A] Average non-glandular trichome density at three stages of leaf development (emergent, young and mature).
DISCUSSION
TRICHOME DISTRIBUTION AND MICROMORPHOLOGY
These raised cells support the trichome (Ascensao et al., 1999) and provide an attachment point that “anchors” the trichome to the epidermal surface (Bhatt et al., 2010a). It is a characteristic feature of trichomes in many plants such as Ceratotheca triloba (Naidoo et al., 2012a), Plectranthus tomentosa (Kim, 2013) and Stachys alopecuros (Venditti et al., 2013a). Leaf surfaces may be covered by both peltate and capitate trichomes, only peltate, only capitate, or in rare cases neither type (Huang et al., 2008).
Both peltate and capitate gland trichomes have been shown to accumulate secretory material in a subcuticular space of the gland secretory head in the family Lamiaceae (Ascensão et al., 1995; Turner et al., 2000; Ventrella and Marinho, 2008; Naidoo et al., 2013). The subcuticular space is formed by the detachment of the cuticle from the cell wall (Ascensao et al., 1999; Tissier, 2012). Release of secretion in gland trichomes can occur via cuticular rupture in vulnerable areas of the secretory head (Jia et al., 2012; Naidoo et al., 2013) through a single apical pore or multiple micropores (Ascensão et al., 1999; Naidoo et al., 2012b) or even by diffusion through the cuticle (Caissard et al., 2012).
Cuticle breakage can also be due to external factors such as high temperature, low humidity or mechanical damage (Ascensao et al., 1995; Ventrella and Marinho, 2008; Baran et al., 2010). A similar phenomenon of cuticular rupture was observed in Plectranthus grandidentatus (Mota et al., 2013) and Teucrium salviastrum (Cavaleiro et al., 2002).
HISTOCHEMICAL AND PHYTOCHEMICAL PROFILE OF S. NATALENSIS
Neck cells with cutinized walls are also present in members of Boraginaceae (Cordia verbenacea, Ventrella and Marinho, 2008), Pedaliaceae (C. triloba, Naidoo et al., 2012a) and other Lamiaceae (S. italica, Giuliani et al., 2011) . The thickened neck cells of the cap trichomes are thought to prevent the apoplastic backflow of exudates whose potentially phytotoxic composition can pose a threat to the rest of the plant if released (Serrato-Valenti et al., 1997; Werker, 2000; Baran et al.). al., 2010). Non-cellulosic polysaccharides such as lignin provide a framework of trichome strength to withstand high mechanical loads (Bischoff et al., 2010).
In a study by Maa et al. 2012) complex carbohydrates were extracted from Stachys floridana and tested for its hepatoprotective activity. Phenolic compounds include, among others, simple phenols, phenolic acids, coumarins, flavonoids and condensed tannins, which are involved in pollination mechanisms, plant pigmentation and plant defense strategies (Blainski et al., 2013). Phenolic compounds present in gland secretory structures may be implicated in plant organ protection against UV-B radiation resulting from exposure to sunlight (Combrinck et al., 2007).
Flavonoids are involved in inflammatory processes and possess antibacterial and antifungal properties (de Sousa-Araujo et al., 2008). In a study on the antioxidant activity of selected Stachys taxa, Bilusic-Vundac et al. 2007) determined that the ethanolic extract of S.
MICROPROPAGATION OF STACHYS NATALENSIS HOCHST
Sodium hypochlorite is a highly effective bactericidal agent, significantly reducing bacterial populations even at micromolar concentrations (Oyebanji et al., 2009: Sen et al., 2013). In a study by Thompson et al. 2009) decontamination of stem tip explants with dilute NaClO (1:3) treatment was not significantly different from a 0.2% HgCl2 treatment. Many studies support the efficacy of different concentrations of BAP in combination with IBA for shoot initiation (Laribi et al., 2012; Chae et al., 2013;.
85 Shoot initiation and proliferation is regulated by the concentration ratio of cytokinin to auxin within the growth medium (Das et al., 2013). Auxins are routinely applied exogenously to encourage lateral and adventitious root formation in tissue culture (Gutierrez et al., 2012; Sevik and Guney, 2013). This combination was previously responsible for the largest percentage of roots in a variety of species such as Hypericum perforatum (Goel et al., 2009).
This has been attributed to its higher stability and persistence in plant tissues compared to IAA (Ludwig-Muller et al., 2005; Nakhooda et al., 2011). Root formation can be induced by any type of auxin, but the mechanism of auxin action at different concentrations and the effect on morphological and physiological development of in vitro plantlets must be considered (Nakhooda et al., 2011).
CONCLUSION
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