In Vitro Selection and Characterisation of Iron-Efficient Potato Cell Lines A
Total Page：16
File Type：pdf, Size：1020Kb
In vitro selection and characterisation of iron-efficient potato cell lines A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy in Biotechnology at the University of Canterbury By Georgina Addae Boamponsem 2016 ABSTRACT Iron (Fe) is an essential micronutrient involved in life-sustaining biochemical processes including photosynthesis, DNA synthesis and repair, antioxidative defence, hormone synthesis and respiration. Fe bioavailability, however, is a major constraint for crop production due to low Fe solubility resulting from multiple soil and /or biochemical stresses. Iron deficiency-induced chlorosis (IDC) is a widespread nutritional disorder in plants worldwide especially in calcareous soils. To reduce the economic impact of Fe-deficiency stress, it has become necessary to deploy in vitro tissue culture as a quick and cost-effective tool for developing stress-tolerant plants. The development of IDC-tolerant (Fe-efficient) plants presents an important means of alleviating IDC. The aim of the present study was to investigate the hypothesis that using in vitro selection, it would be possible to exploit somaclonal variation and the inherent ability of plants to evolve adaptive mechanisms under Fe-deficiency stress for the generation of novel Fe-efficient potato plant lines. To achieve this, an in vitro selection strategy was designed to generate putative Fe-efficient potato (Solanum tuberosum cv ‘Iwa’) cell lines and plants were regenerated from these cell lines. The Fe-efficient plant lines derived from these cell lines were characterised, using various morphological, biochemical and molecular parameters, regarding their responses to Fe- deficiency under in vitro conditions. A two‐step direct selection scheme employing Fe deficiency (0-5 µM) as selective pressure was applied in generating novel Fe-efficient somaclonal variants. Fe-efficient callus cells were selected on half-strength Murashige and Skoog (MS) medium supplemented with 1.78 µM N6-benzylaminopurine (BA), 3.22 µM α-naphthaleneacetic acid (NAA) and 0-5 µM FeNaEDTA. Shoot buds were regenerated from the calli exhibiting IDC tolerance when cultured on half-strength MS medium supplemented with 6.66 µM BA and 2.89 µM gibberellic acid (GA3) and 35 plant lines were established. The IDC tolerance of the 35 plant lines was confirmed when they were grown on plant growth regulator-free medium supplemented with 0-5 µM FeNaEDTA. Using IDC scores as the evaluation criteria, 23% of the plant lines (8) could be regarded as Fe-efficient and 77% (27) as Fe-inefficient plant lines. The compact potato callus cultures produced were used to investigate more closely their responses to a short-term (1 month) and an extended period (3 months) of a Fe deficit or sufficiency in the culture medium. The morphological responses include visual chlorotic symptoms (yellowing), reduced fresh weight and area of growth covered by calli grown on Fe-deficient medium. A Fe deficit in the medium led to decreases in chlorophyll and I carotenoid contents, reduction in activities of peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX) enzymes accompanied with an increase in lipid peroxidation in calli. Exposure of calli to Fe deficiency enhanced ferric chelate reductase (FCR) activity, induced phenolic production and increased hydrogen peroxide (H2O2) generation. Histochemical staining of Fe showed that whereas Fe distribution in cells of calli cultured on Fe-deficient medium was sparse, Fe was widely distributed among actively dividing callus cells cultured on Fe-sufficient medium. The morphological and biochemical responses assessed were pronounced with prolonged exposure to Fe deficiency leading to severe chlorosis and/or death of cells in chlorosis-susceptible calli but chlorosis-tolerant calli cells maintained their greenness and viability. These findings have contributed to a better understanding of Fe nutrition in potato at the cellular level. The morphological, biochemical and molecular mechanisms conferring differential tolerance to Fe deficiency in two contrasting groups of potato plant lines established in this study were characterised. Fe-inefficient (INF) plant lines and control plants exhibited severe chlorosis when grown on a Fe-limiting medium, but Fe-efficient (EF) lines were tolerant to chlorosis. The EF plant lines were of shorter statures as far as stem height, root length, internodal distances were concerned compared to INF and control plants. Formation of lateral roots and root hairs was enhanced in EF plant lines compared to INF and control plants, suggesting that these might be morphological adaptations in the root of the EF plant lines in response to a deficit in Fe supply. There was a significantly positive correlation between chlorosis score and root length in control plants and INF plant lines, indicating that root length could contribute to IDC susceptibility. Furthermore, stem height was found to have a highly positive relationship with intermodal distance, leaf and root lengths in EF plant lines. Biochemically, IDC tolerance was linked to increased chlorophyll contents, FCR and POD enzyme activities in the leaves of EF plant lines. The absence of a similar adaptive strategy in leaves of INF lines could underpin their susceptibility to Fe deficiency conditions. FCR and POD activities in the roots of EF plant lines increased greatly while chlorophyll content decreased. The reverse was observed in the roots of INF plant lines, implying that the aforementioned responses contributed to Fe-efficiency at the root level. Increased POD activities in both roots and leaves of EF plant lines suggest that improved capacity to detoxify ROS plays a role to adapt to Fe deficiency stress. Leaf carotenoid content was highly variable among EF and INF plant lines but the carotenoid content decreased in the roots of EF compared to INF plant lines. It would seem that carotenoid content in the roots rather than the leaves might be a more suitable indicator for assessing differential tolerance to IDC. Fe II deficiency resulted in the reduction in phenolic concentration in roots and leaves of IFN and some EF plant lines relative to control plants. However, 40-50% of the EF lines (A1, B2, E1- 3) showed higher amounts of phenolics in roots and/or leaves. The findings suggest that IDC tolerance is related to improvement in ferric reductase ability, regulation of chlorophyll biosynthesis and enhancement of the antioxidant potentials. These parameters may serve as predictors for Fe-efficiency trait in plants. Transcriptional responses of both sets of potato plant lines provide evidence that ferritin (FER3) and the iron-regulated transporter (IRT1) play a role in the acquisition of iron from sources with low bioavailability mainly in the leaves and roots respectively. The A1 chlorosis-tolerant plant line exhibited characteristic Fe-efficiency responses of increased fer3 and irt1 transcripts in leaf and root organs. Significantly increased fer3 expression was detected in the leaves of a higher proportion (62%) of putative Fe-efficient lines than in roots (37.5%). IRT1 expression level was 20% significantly greater in roots than in leaves. IRT1 transcripts in the roots of 50% of the potential EF plant lines (A1, B2, B9, E3) was enhanced in response to Fe deficiency. Some IFN plants had higher values of gene expression compared to EF plants. In conclusion, a novel set of Fe-efficient and inefficient potato plant lines has been developed which can be useful in further research and breeding programs aiming at selecting IDC tolerant potato genotypes. Taken together, the results showed that 37.5% of the putative EF plant lines (A1, B2, B9) have both the capacity to elicit improved morphological and biochemical characteristics in responses to Fe-deficiency stress as well as to enhance the expression of Fe homeostasis-related genes. The in vitro selection approach used in this study can be applied for the selection of Fe-efficiency in other plant species. III DEDICATION I dedicate this research to God Almighty, my parents (Grace Danka and James Addae), my husband (Dr. Louis Boamponsem) and children (Baruch and Theresa). IV ACKNOWLEDGEMENTS I am extremely grateful and appreciate the support of my supervisors, Prof. David Leung and Dr. Carolyn Lister without whom, this work could never have become a reality. Their encouragement, patience and enthusiasm gave me room to explore, take initiatives and to develop. Their laughter and kindness will always be cherished. Sincere thanks to the School of Biological Sciences, especially the technical staff: Graeme Bull, Manfred Ingerfeld, Rayleen Fredericks, Craig Galilee, Jackie Healy, Thomas Evans and Reijel Gardiner. Many thanks to Nicole Lauren-Manuera (MPI training) and Mattew Walters (posters and photography). They were always knowledgeable, patient and caring. I am very grateful to my lab mates and friends (Keum-Ah Lee, Seyedardalan Ashrafzadeh, Solomon Wante and Gloria Adjapong) for their time, friendship and assistance in various ways. Enormous thanks to Matthew Van Voorthuizen and Dr. Pragathi Dhandapani for all their support in enabling me to work comfortably with RT-PCR equipment and systems. A very special thank you to my husband, Dr. Louis Boamponsem, for his continued support, reassurance and technical advice on statistics. I am grateful to my children, Baruch and Theresa for spicing up my life with giggles and happiness. They always give me something refreshing to look up to and help me appreciate calmness