Thesis (MTech (Horticulture)--Cape Peninsula University of Technology, 2017. / A study was conducted to investigate the effects of various media composition and wounding treating on the in vitro propagation of Argyroderma subalbum and A. testiculare explants derived from mature plants, antioxidants and plant growth regulators (PGR) concentrations. One experiment consisted of 3 medium types including Murashige and Skoog (MS) medium strength, vitamin supplement. Fifteen replicates were used for each treatment. The shoots were then sub-cultured to ten replicate regenerated medium consisting of varying levels and combination of indole-3-acetic acid (IAA) and 10 μM 6-Benzyladenine (BA) supplements. In another experiment consisted of varying levels of auxins with MS medium strength, activated charcoal (AC) and vitamin supplements ten replicates were used for each treatment. Results indicated the positive role of cytokinins types’ 6-Benzyladenine (BA), 2-isopentyladenine (2iP) and Kinetin in inducing callus formation from wounded explants. The highest rate of friable callus formation of wounded explants was observed in media containing vitamin supplementation with BA at 10 μM. Callus formation significantly increased with the addition of vitamins at 10 μM on BA, 2iP and kinetin. With regards to the effects of various media composition and wounding explants on in vitro growth and regeneration of A. subalbum and A. testiculare, significant results were achieved with BA, 2iP and kinetin concentrations on explants discoloration and callus formation. The antioxidant treatment, AC did not reduce explants discoloration, but the induction of the callus was developed furthermore, results showed that IAA with BA concentrations without addition of AC there was significantly difference on both species but A. subalbum dominated with browning intensity (Chapter 3). Only sub-culturing of the explants succeeded in preventing explants discoloration and subsequently increased the number of shoots. The interaction between Indole-3-acetic acid (IAA) concentrations combined with BA resulted in the most effective technique in reducing explants discoloration at the media contact point. This study provides an insight into the contributing factor and methods of overcoming the major problem of phenolic oxidation and promoting the in vitro growth and regeneration of A. subalbum and A. testiculare.

No abstract available. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2003.

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Cauliflower (Brassica oleracea var. botrytis) is described as a recalcitrant plant to genetic transformation processes especially Agrobacterium-mediated and as an extremely low frequency event then it requires a large amount of explants for this procedure to succeed. This thesis describes the development and refinement of a mass propagation system for cauliflower micropropagation and its use for overcoming recalcitrance to genetic transformation. Shoot meristematic tissue was taken from the curd of cauliflower and used to establish in-vitro cultures in liquid medium. Explants were cultured in a Murashige and Skoog (MS) medium containing various plant growth regulators combinations to induce shoot regeneration and which were optimised to be 2 mg L-1 (9.29 μM) kinetin and 1 mg L-1(4.9 μM) IBA. Shoots were cultured for 4–6 weeks to obtain rooted plants, which were then suitable for weaning and subsequently produce fully- developed in-vivo plants in pots in soil with a 95%+ success rate. A procedure for detection of the presence of insert DNA in recombinant plasmids in individual Agrobacterium tumefaciens strains was refined. Cauliflower was transformed using the EHA105 strain of A. tumefaciens harboring the binary vector pPRTL2 plasmid carrying the antioxidant gene Ascorbate peroxidase (APX) for increased stress resistance coupled with neomycin phosphotransferase II (nptII) for resistance to kanamycin and β-glucuronidase (GUS) as a marker gene. Selection was carried out in MS medium containing kanamycin (50 mg L-1), and surviving tissues were then tested by histochemical GUS assay.Agrobacterium-mediated plant genetic transformation requires a two-step process for its success: selection and regeneration of transformed tissues, and the elimination of the transformation vector (Agrobacterium). This study used carbenicillin and cefotaxime in MS media to eliminate A. tumefaciens, at selection levels of 25 and 50 mg L-1 kanamycin. Kanamycin severely reduced explant growth and regeneration of control cultures at concentrations as low as 10 mg L-1 and completely inhibited shoot organogenesis at 50 mg L-1. The integration of APX gene into putative transformant lines was confirmed using GUS and leaf disc assays. Genomic integration of the gene cassette was optimised using PCR analysis with primers flanking npt II and CaMV promoter regions. The stable integration of the APX gene in the putative transgenic plants was detected using PCR at 478bp. The result confirmed the first report of transformation with APX gene in Brassica oleracea. Thus, a protocol for effective Agrobacterium-mediated genetic transformation of cauliflower was optimized. Transformed and control lines were sub-cultured many times on maintenance medium over 2 years without any loss of the transgene and then tested for salt resistance as in-vitro and in-vivo plants using a leaf disc assay. Control plants had little or no NaCI resistance whilst transformed plants showed varying degrees of resistance. Analysis of APX gene expression under salt treatment showed that putative transgenic cauliflower survived salinity stress compared with control plants. Non-acclimated and acclimated in-vivo plants were also assessed for resistance to frost. Both non-acclimated and acclimated APX transformed lines showed improved frost resistance compared to controls. The results clearly confirmed that NaCI and frost resistance were stable traits attributable to improved APX expression.

M.Tech. / Sorghum [Sorghum bicolor (L.)] is classified as a relatively recalcitrant crop due to its poor amenability to in vitro and genetic manipulation. An efficient and reproducible in vitro plant regeneration method is vital for a successful transformation of any crop. Plant regeneration and transformation of eight selected elite sorghum genotypes was studied. Immature zygotic embryos were used as explants and cultured on two different callus induction media. Three genotypes ICSV1111N, SRN39 and P898012 were found to be highly regenerable producing 5.99; 5.1 and 4.74 regenerants per explant respectively on the G2+L-proline callus induction medium. The eight elite sorghum genotypes were co-bombarded with the uidA reporter gene and manA selectable marker gene. Bombarded immature zygotic embryos were selected on G2+L-proline callus induction medium supplemented with mannose as a selective agent. PCR Positive transformants were only obtained from genotype P898012. Furthermore the genotype P898012 was stably transformed with a lower DNA amount of manA minimal transgene. The manA gene presence was confirmed with PCR and southern blot analyses and a transformation efficiency of 0.38% was attained. The fertile transgenic plants displayed simple integration patterns, and the gene was also inherited to the T1 progeny of manA resistant trasnsformants in a Mendelian fashion.

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In vitro culture techniques are widely used in potato for rapid production of specific pathogen tested plantlets and microtubers, which are essential for seed certification programs. The limited calcium (Ca) uptake in vitro promotes physiological disorders in many micropropagated species including potato. Factors that may affect Ca uptake by potato plantlets, including cations, osmotic potential, and boron (B) were examined. The possibility of improving microtuber yield by low pH shock or successive harvests, which stimulated greenhouse potato production, were also investigated. Ca uptake by cvs. Norland and Bintje potato plantlets grown on modified Murashige and Skoog (MS, 1962) media was enhanced by diluting the total macroelements in the nutrient medium, except Ca. Increasing Ca:specific cation ratios, by lowering either NH 4, Mg or K, had little effect on Ca uptake by Norland plantlets. At equivalent Ca:total cation, Ca uptake was promoted further when the total macroelement concentration was reduced by half than when the Ca level was doubled (from 3 to 6 mM). This suggested that the increase in the osmotic potential of the medium had a great impact on Ca uptake, probably by affecting the root pressure. B contamination from impurities in the nutrient chemical components always occurred. The addition of the recommended H3BO 3 level of 0.1 mM (1.08 ppm) or more (0.3 mM) to the medium decreased Ca content of leaves and shoots in Norland but not Bintje, while addition of only 25% of this level was sufficient to provide plantlets with adequate B tissue concentration without compromising normal growth. Macroelement dilution along with Ca addition, and lower B levels than commonly used in MS (0.1 mM) are recommended for optimal Ca uptake in potato and possibly other plant species. Microtuber yield was not improved by a low pH shock. Adjusting microtuberization medium pH to less than 5.7 (3.5, 4.3 or 5.2), prior to autoclaving, reduced microtuber number and/or weight