Browsing by Author "Nakhooda, Muhammad."

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The actions of, and interactions between, auxins and cytokinins and their effect on in vitro rooting of selected Eucalyptus clones.
(2011) Nakhooda, Muhammad.; Watt, Maria Paula Mousaco Deoliveira.; Mycock, David John.
Clonal propagation of Eucalyptus spp. and its hybrids allows for competitiveness in the commercial forestry industry through the propagation and preservation of superior/elite genotypes. Vegetative propagation through rooted cuttings is the industry‟s standard and the choice of clones selected for plantations are determined by their rooting ability. However, as many potentially valuable genotypes are recalcitrant to adventitious rooting, micropropagation is the only effective means of propagating them. Micropropagation results in high plantlet yields, achieved primarily through the empirical use of the key plant growth regulators (PGRs) cytokinins and auxins, for shoot and root production, respectively. Their selection for use in vitro is driven by their effects on percent rooting rather than root quality. Little is known regarding the quality of the roots of the plantlets ex vitro, but there is some evidence that they are different from those of seedlings and cuttings. It was therefore hypothesized that the properties of exogenous PGRs and their interaction with other exogenous and endogenous PGRs, influenced root development and subsequent root quality. This was tested in vitro using a good-rooting E. grandis (TAG31) and two poor-rooting E. grandis x nitens hybrid clones (GN155 and NH58). In the former, the auxins supplied during the pre-rooting culture stages (multiplication and elongation) were sufficient for 100% rooting in an auxin-free rooting medium. Different combinations of PGRs in the two pre-rooting stages, followed by rooting without auxins, revealed a direct relationship between the stability of the supplied auxin and the rooting ability of TAG31. Gas chromatographymass spectrometry (GC-MS) analyses indicated that endogenous shoot levels of indole- 3-acetic acid (IAA) influenced graviperception. Also, low IAA content was associated with atypical starch grain accumulation or its absence from root tips (53.1 nmol IAA gˉ¹ DW compared with 325.7 nmol IAA g-¹ DW in gravisensing roots). The specific roles of the natural auxins IAA and IBA on root morphogenesis were then investigated using 2,3,5-triiodobenzoic acid (TIBA; inhibits IAA transport), ρ-chlorophenoxyisobutyric acid (PCIB; inhibits auxin signal transduction), and the auxin antagonist kinetin in the rooting medium, following root induction. After 3 weeks, the mean root diameter was significantly reduced from 552.8μm (control) to 129.2μm (with PCIB) and 278.6μm (with kinetin). TIBA increased root diameter to 833.4μm, decreased Δ root length, increased root vasculature and resulted in agravitropism. Hence, whereas rooting could be induced by IBA, IAA was necessary for the maintenance of vascular integrity and graviperception. This critical role of IAA in root development is of importance as IBA, owing to its higher stability, has been traditionally relied upon for root induction in the majority of micropropagation protocols. The potential of incorporating IAA into the media formulations of in vitro protocols for poor-rooters that do not respond well to IAA was then investigated, using GN155 and NH58. While PCIB in the rooting medium of GN155 completely inhibited rooting, the addition of dihydroxyacetophenone (DHAP), an inhibitor of auxin conjugation, to the rooting medium, did not significantly increase % rooting in the presence of 0.1 mg 1ˉ¹ IBA (i.e. 50% rooting with 2mM DHAP and IBA, compared with 45% with IBA alone). The results suggested that the inability of some eucalypts to induce roots easily in vitro was not due to a deficiency in auxin signal transduction or to auxin conjugation. Instead, rooting was inhibited by an accumulation of kinetin within shoots during the pre-rooting culture stages. The endogenous levels of PGRs in shoots of GN155 and NH58 showed a strong relationship (R² = 0.943) between the shoot kinetin:auxin and shoot rootability. Substituting kinetin with the relatively less stable natural cytokinin trans-zeatin in the elongation stage resulted in a significant increase in % rooting in both clones, from 19% to 45% (GN155) and from 31% to 52% (NH58), with 0.1 mg 1ˉ¹ IAA in the rooting medium. However, omitting all cytokinins from the elongation medium, resulted in over 95% and 75% rooting of shoots of GN155 and NH58, respectively, with 0.1 mg 1ˉ¹ IAA. These results suggest that IAA is a requirement for root development and cannot be substituted by its analogues in certain root developmental events. Hence, IAA should be the preferred auxin for eucalypt micropropagation. As fundamental research, the approach taken in this study circumvents the empirical method used in improving micropropagation protocols. The importance of the properties and the interactions between endogenous and exogenous PGRs in regulating root morphogenesis, and the practical implications of these findings is emphasised.
Micromorphology and phytochemistry of the foliar secretory structures of Stachys natalensis Hochst. and development of an in vitro propagation protocol.
(2014) Kalicharan, Benita.; Naidoo, Yougasphree.; Nakhooda, Muhammad.
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 uni- or 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. 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 superior-selected genotypes, and thus potentially maximising the yield of putative pharmacologically-important secondary metabolites.
Survival and rooting of selected vegetatively propagated Eucalyptus clones in relation to supplied auxin.
(2013) Rambaran, Natasha.; Watt, Maria Paula Mousaco Deoliveira.; Mokotedi, Mompe Edward Oscar.; Nakhooda, Muhammad.
Eucalyptus spp. and hybrids dominate the global plantation forestry industry, and vegetative propagation through cuttings is the preferred method for their commercial use. However, the cuttings of some species and hybrids show recalcitrance to rooting. The first aim of this study was to improve percentage rooting of three clones of E. grandis x E. nitens (Clones 1, 2 and 3) identified by a commercial nursery as having variable rooting abilities. The second was to relate their rooting responses as cuttings to their rooting responses in vitro. Minicuttings (3.5 – 4 cm in length) (hereafter referred to as cuttings) were subjected to commercial nursery propagation practices. Initial results revealed that in the absence of exogenous plant growth regulators (PGRs), soft (juvenile, thin diameter) cuttings survived (87 – 95%) and rooted (29 – 32%) significantly better than hard (mature, thick diameter) ones (62 – 71% survival and 2 – 8% rooting). This validated the use of soft cuttings by the nursery and all subsequent studies were conducted with soft cuttings. The other nursery practice of applying the commercial rooting powder Seradix 2 (3 g kgˉ¹ indole-3-butyric acid [IBA]) adversely affected the survival and subsequent rooting of cuttings of Clones 1 and 2. Ensuing studies investigated: 1) the effect of mode of IBA application (powder vs. liquid); 2) concentrations of Seradix (0, 0.5, 1, 2 and 3 g kgˉ¹ IBA), applied at initial placement of cuttings and two weeks later; and 3) the influence of season on the survival and subsequent rooting of cuttings. Results showed that regardless of the mode of application, IBA significantly reduced percentage survival and rooting in cuttings of Clones 1 and 2. The delayed application of Seradix, two weeks after cuttings were initially set, resulted in a higher percentage survival and rooting than when cuttings were supplied with Seradix at initial placement. Nevertheless, the best survival for Clones 1, 2 and 3 (95%, 99% and 71%, respectively) and rooting (83%, 64% and 47%, respectively) occurred in the absence of Seradix. In addition, the survival and rooting of cuttings were seasonally variable, with particularly low rooting during winter (e.g. for Clone 1, 32%) when compared with summer (e.g. for Clone 1, 83%). Shoots from all the clones were multiplied in vitro, followed by elongation on either of two media (E1= kinetin, α-naphthalene acetic acid [NAA] and IBA; E2 = kinetin and indole-3-acetic acid [IAA]), and then rooting on 0, 0.1 or 1.0 mg 1ˉ¹ IBA. The latter were selected to typify the range of Seradix concentrations used for the cuttings (i.e. no IBA, low and high IBA concentrations). For all three clones, shoots elongated on E1 or E2 displayed high survival (> 80%) but failed to root without IBA in the rooting medium. For Clones 1, 2 and 3 the best in vitro survival (80%, 100% and 100%, respectively) and rooting (40%, 75% and 40%, respectively) occurred when shoots were elongated on E2 and rooted on 0.1 mg 1ˉ¹ IBA. However, 1.0 mg 1ˉ¹ IBA in the rooting medium severely inhibited survival (0 – 50%), irrespective of the clone or the elongation treatment used. Overall, cuttings demonstrated the best survival and rooting in the absence of exogenous IBA, which suggested that sufficient endogenous auxin was present within the shoots for successful root induction. The application of exogenous IBA may have disrupted the cuttings’ endogenous PGR balance resulting in an inhibition of survival and rooting. In vitro shoots required a low concentration of IBA (0.1 mg 1ˉ¹) in order to counteract the antagonistic effect of cytokinins that were supplied during the multiplication and elongation culture stages, and promote rhizogenesis. Essentially, both cuttings and in vitro shoots demonstrated adverse survival and rooting responses when subjected to excessively high IBA concentrations.