Growth medium and environmental studies of sweet potato meristem culture : a thesis presented in partial fulfilment of the requirements for the degree of Master in Applied Science at Massey University, New Zealand

The ability of three New Zealand local sweet potato (Ipomoea batatas L.) cultivars 'Toka Toka Gold', 'Beauregard', and Owairaka Red' to form plantlets in vitro was investigated Meristematic tips (0.2–0.4 mm) of apical shoots from vines of the three cultivars, and from tubers of 'Owairaka Red' were cultured in modified Murashige and Skoog (1962) medium (MS medium) containing plant growth regulator (s). Cultivars and organs of explants differed in response to exogenous levels of plant growth regulator(s) and in the rate of proliferation. Optimal regeneration occurred in liquid MS medium supplemented with BA 0.1 mg/1 for 'Toka Toka Gold' and Owairaka Red' (from vines), and with BA 0.5 + IBA 0.1 mg/1 for 'Beauregard'. For Owairaka Red' (from tubers), MS liquid medium with BA 0.3 mg/1, and MS liquid medium with GA3 20 mg/1 (plus other organic compounds) proliferated shoots and plantlets. Continuous lighting inhibited the proliferation of plantlets in all three cultivars. Regeneration was strongly affected by the age of the shoots from which the explants were excised and the season when cultures were begun. Successful culture was obtained by culturing explants from young shoots in the Spring

STUDIES ON KOLA TISSUE CULTURE I: Protocols for Establishing Kola Tissues in vitro

The micropropagation of Cola nitida (Vent.) Schott and Endlicher by means of tissue culture was investigated to provide baseline infonnation on the requ irements for the survival of kola tissues and organs in vitro. Investigations were conducted on development of sterilization protocols, and medium selection and modification. The best sterilization procedure was established with the step-wise treatment of 70% ethanol, for 20 seconds and 10% (w/v) CaOCl, for I 0 minutes .. The use of modified Murashige-Skoog (MS) medium (without Zn and Cu elements) as basal medium was found as appropriate as the original MS medium, for explants' survival. The appropriate antioxidant technique was also established with 10mgl-1 ascorbic acid

Regeneration ability and genetic transformation of root type chicory (Cichorium intybus var. sativum)

To develop an efficient protocol for shoot regeneration of root chicory (Cichorium intybus var. sativum), some factors, including different concentrations of plant growth regulators in Murashige and Skoog (MS) medium, type of explants and genotypes were evaluated. Initiation of callusing were best achieved in MS medium supplemented with 1-naphthaleneacetic acid (NAA) (0.1 mg l-1) plus 6-Benzylaminopurine (6-BAP) (1 mg l-1), indole-3-acetic acid (IAA) (0.01 mg l-1) plus 6-BAP (1.0 mg l-1), and IAA (0.5 mg l-1) plus (0.5 mg l-1) 6-BAP combinations on leaf and cotyledon explants. Explant-derived calli were able to produce multiple adventitious shoots in MS medium containing IAA (0.5 mg l-1) plus 6-BAP (0.5 mg l-1). MS medium containing indole-3-butylric acid IBA (1 mgl-1) efficiently induced rooting on elongated shoots. Various responses to the number of generated shoots were observed when regeneration abilities of different chicory cultivars were examined. Among root and “Witloof” cultivars, ‘Melci’ and ‘Hera’ belong to the root cultivars and exhibited higher shoot regeneration ability. Using the optimized regeneration method, genetic transformation of ‘Melci’ with Agrobacterium tumefaciens strain C58C1 RifR (pGV2260) (pTJK136) was successfully carried out. Histochemical GUS assay, polymerase chain reaction (PCR) and reverse transcription-polymerase chain reaction (RT-PCR) analysis of putative transformed plants confirmed successful integration of the T-DNA into the chicory genome. Expression of the neomycine phosphotransferase (NPTII) in the regenerated plants was also shown by well-developed roots on root inducing medium containing 100 mg l-1 kanamycin. This simple, efficient and reproducible protocol could be useful for inducing somaclonal variation and genetic modification of root chicory cultivars to broaden genetic variation and transferring of important genes

Thidiazuron-induced shoot organogenesis from mature leaf explants of scented Pelargonium capitatum cultivars

Shoot organogenesis from mature leaf tissues of two scented Pelargonium capitatum cultivars, ‘Attar of Roses’ and ‘Atomic Snowflake’, grown in the greenhouse, were optimized in the presence of thidiazuron (TDZ). The protocol involved preculture of leaf sections on basal Murashige and Skoog (MS) medium supplemented with 10 lM TDZ, 4.4 lM of 6-benzyladenine (BA) and 5.4 lM a-naphtaleneacetic acid (NAA) for a period of 2 weeks and followed by subculture of explants to a fresh medium containing 4.4 lM BA and 5.4 lM NAA. Frequency of regeneration reached approximately 93% for both cultivars, with the induction of more than 100 shoots per explant. Regenerated plantlets were rooted on half-strength MS medium supplemented with 4.4 mM sucrose and 8.6 lM of Indole-3-acetic acid (IAA). All regenerated shoots from both cultivars developed roots when transferred to organic soil mix, acclimatized, and successfully transferred to greenhouse conditions. When regenerated shoots were transferred to hydroponic conditions, frequency of survival was 76.2 and 61.9% for ‘Attar of Roses’ and ‘Atomic Snowflake’, respectively

Proteomics as a quality control tool of pharmaceutical probiotic bacterial lysate products

Probiotic bacteria have a wide range of applications in veterinary and human therapeutics. Inactivated probiotics are complex samples and quality control (QC) should measure as many molecular features as possible. Capillary electrophoresis coupled to mass spectrometry (CE/MS) has been used as a multidimensional and high throughput method for the identification and validation of biomarkers of disease in complex biological samples such as biofluids. In this study we evaluate the suitability of CE/MS to measure the consistency of different lots of the probiotic formulation Pro-Symbioflor which is a bacterial lysate of heat-inactivated Escherichia coli and Enterococcus faecalis. Over 5000 peptides were detected by CE/MS in 5 different lots of the bacterial lysate and in a sample of culture medium. 71 to 75% of the total peptide content was identical in all lots. This percentage increased to 87–89% when allowing the absence of a peptide in one of the 5 samples. These results, based on over 2000 peptides, suggest high similarity of the 5 different lots. Sequence analysis identified peptides of both E. coli and E. faecalis and peptides originating from the culture medium, thus confirming the presence of the strains in the formulation. Ontology analysis suggested that the majority of the peptides identified for E. coli originated from the cell membrane or the fimbrium, while peptides identified for E. faecalis were enriched for peptides originating from the cytoplasm. The bacterial lysate peptides as a whole are recognised as highly conserved molecular patterns by the innate immune system as microbe associated molecular pattern (MAMP). Sequence analysis also identified the presence of soybean, yeast and casein protein fragments that are part of the formulation of the culture medium. In conclusion CE/MS seems an appropriate QC tool to analyze complex biological products such as inactivated probiotic formulations and allows determining the similarity between lots

STUDIES ON KOLA TISSUE CULTURE II: Effect of plant growth regulators on callus induction

Callus induction was studied on leaf and single node cutting explants obtained from Cola nitida (Vent.) Schott and Endlicher seedlings. Callus was induced successfully from cut surfaces (periphery) ofyoung leaves in vitro on the medium supplemented with 0.5-5.0 mgl-1 naphtheneacetic acid (NAA), combined with 0.23 mgt- I 6-benzyl-aminopurine (BAP) and on medium supplemented with 0.2-0.6 mgl-1 BAP, combined with 1.0 mgl-1 NAA. Successful callus induction was al so obtained from the buds of single nodal explants cultured on MS medium supplemented with 0.1-1 .0 mgl-1 NAA, combined with 2.3 mgl-1 BAP

Metabolic perturbations associated with the consumption of a ketogenic medium-chain TAG diet in dogs with idiopathic epilepsy

Consumption of diets containing medium-chain TAG (MCT) has been shown to confer neuroprotective effects. We aim to identify the global metabolic perturbations associated with consumption of a ketogenic diet (medium-chain TAG diet (MCTD)) in dogs with idiopathic epilepsy. We used ultra-performance liquid chromatography-MS (UPLC-MS) to generate metabolic and lipidomic profiles of fasted canine serum and made comparisons between the MCTD and standardised placebo diet phases. We identified metabolites that differed significantly between diet phases using metabolite fragmentation profiles generated by tandem MS (UPLC–MS/MS). Consumption of the MCTD resulted in significant differences in serum metabolic profiles when compared with the placebo diet, where sixteen altered lipid metabolites were identified. Consumption of the MCTD resulted in reduced abundances of palmitoylcarnitine, octadecenoylcarnitine, stearoylcarnitine and significant changes, both reduced and increased abundances, of phosphatidylcholine (PC) metabolites. There was a significant increase in abundance of the saturated C17 : 0 fatty acyl moieties during the MCTD phase. Lysophosphatidylcholine (17 : 0) (P=0·01) and PC (17:0/20:4) (P=0·03) were both significantly higher in abundance during the MCTD. The data presented in this study highlight global changes in lipid metabolism, and, of particular interest, in the C17 : 0 moieties, as a result of MCT consumption. Elucidating the global metabolic response of MCT consumption will not only improve the administration of current ketogenic diets for neurological disease models but also provides new avenues for research to develop better diet therapies with improved neuroprotective efficacies. Future studies should clarify the involvement and importance of C17 : 0 moieties in endogenous MCT metabolic pathways