Nurashikin Kemat, Nurashikin Kemat (2020) Improving the quality of tissue-cultured plants by fixing the problems related to an inadequate water balance, hyperhydricity. Doctoral thesis, Wageningen University.
![[img]](https://repositori.mohe.gov.my/style/images/fileicons/text.png) |
Text
Improving the quality of tissue-cultured plants by fixing the problems related to an inadequate water balance, hyperhydricity.pdf Restricted to Registered users only Download (7MB) |
Abstract
In vitro tissue culture is a technique for producing amongst others high numbers of uniform plants. This technique is excellent for accelerating plant propagation and establishment and supplying high quality starting material which can have a positive impact on product commercialization. Also for breeding purposes tissue culture techniques are indispensable for instance for the establishment of plants from embryos rescued from intra- or interspecific crosses or for the production of doubled haploids and genetically modified plants after transformation. Several obstacles may occur during the culture process, one of which is hyperhydricity (HH). HH is a serious problem since it can affect shoot development, the survival and the quality of micropropagated plants after transfer to ex vitro conditions. Hyperhydric shoots are characteri ed by a high water content which causes several morphological abnormalities e.g. curled, brittle, wrinkled, elongated petioles and/or translucent leaves. Hyperhydric shoots can display a range of changed anatomical characteristics and several types of abnormal structures can be recognized. Understanding the underlying mechanisms and factors involved in the control of plant growth in vitro can greatly improve the quality of micropropagated plants. In the present study, quantitative and qualitative physiological, anatomical and biochemical analyses were carried out in normal and hyperhydric Arabidopsis thaliana and Limonium sinuatum (Statice) in vitro grown plants. To elucidate factors which may regulate the occurrence of HH, the effects of medium components like cytokinin (CK) and gelling agents were evaluated using two different Arabidopsis thaliana ecotypes (Col-0 and Ler; Chapter 2). Our results indicated that CK concentration and type and gelrite-solidified medium induced HH. The effects of these were more accumulation of water in the apoplast which caused stomatal closure, higher water retention capacity and declining transpiration in the seedlings compared to their corresponding controls. We found that the application of CK to the medium increased the chance of HH development, with the phenylurea-type CK being worse than the adenine-type CK. Furthermore, we looked at histological and ultrastructural patterns of hyperhydric leaves to identify the changes promoted by this phenomenon and found a disorganized cellular organization and large intercellular spaces in the HH leaves. The water availability of the medium was changed by lowering or raising the concentration of gelling agent and this was found to be of great influence on the level of HH. The results showed that media components were one of the most critical factors of HH. No significant differences in HH symptoms or development were observed between the two Arabidopsis thaliana ecotypes Observations of stomatal closure and apoplast flooding in previous studies prompted us to investigate whether these processes are linked and which of them comes first and might be the true cause of HH in Arabidopsis (Chapter 3). We evaluated the response patterns of stomatal characteristics by examining the effect of plant growth regulators such as the stomatal aperture enhancers, 5-Aminolevulinic acid (ALA) and coronatine, and the stomatal closure inducer salicylic acid (SA) as well as by using mutants affected in stomatal characteristics and transpiration rate. Exogenously applied ALA significantly inhibited stomatal closure and decreased the symptoms of HH. However, another enhancer of stomatal opening, coronatine, caused extensive anthocyanin formation in the seedlings as an indication for enhanced stress and the leaves of plantlets treated with this compound were found to accumulate more apoplastic water, even while stomata were still open. SA application as a compound known to induce stomatal closure, resulted in severe hyperhydric seedlings. Combining ALA together with CK showed HH symptoms and apoplast flooding, accompanied by stomatal closure and a decline in transpiration rate, albeit to a lesser extent than on gelrite alone. We could confirm these findings in the Arabidopsis stomatal density mutants, epidermal patterning factor1 (epf1) and four lips (flp) and found HH symptoms on the seedlings of these mutants even on Micro-agar. Taken together, this indicated that stomatal closure was not the causal factor of HH but when observed, it was a consequence of apoplast flooding leading to HH. In order to determine the fundamental reason for the occurrence of the HH phenomenon and to investigate the hypothesis that CK could affect the characteristics of the cell walls bordering the intercellular spaces, thus influencing HH, we exogenously applied p-coumaric acid, a hydroxyl derivative of cinnamic acid and a precursor for lignin and flavonoids, to gelrite medium (Chapter 4). Exogenously applied p-coumaric acid to Arabidopsis thaliana wild-types, Ler and Col-0, led to increases in apoplastic air and lowering of apoplastic water in seedlings grown on medium solidified with gelrite. Application of p-coumaric acid on Arabidopsis less-lignin-mutants ref 3-3 and ref 3-1 resulted in the same pattern; more apoplastic air and less water which confirmed the role of lignin in HH. Moreover, the results revealed that exogenously applied p-coumaric acid inhibited root growth and increased the total lignin content in both wild-types and less-lignin mutants. We conducted additional experiments to mimic the C4H mutants (ref3) by culturing control seedlings with piperonylic acid (PIP, a specific inhibitor of C4H) on Micro-agar medium in order to prove that lignin plays a central role in HH. The results showed that the seedlings developed HH symptoms accompanied by significant increases in the percentage of apoplastic water and concurrent decreases in the percentage of apoplastic air. In fact, the seedlings after PIP treatment also demonstrated a reduction in total lignin when compared to the control treatment without PIP. Cellular disorganization and cell wall defects were observed in transverse sections of Arabidopsis less-lignin-mutant and Arabidopsis seedlings treated with PIP. Based on our results, we postulate that these cellular deficiencies with large intercellular spaces (ICs) and little lignin deposition are responsible for rendering the apoplast more hydrophilic and together with the effect of higher root pressure and high relative humidity in the headspace eventually cause HH. Increasing lignin content appeared to diminish the symptoms of HH and suggests an important role for lignin in HH. In Chapter 5, we determined the effect of calcium (Ca2+) in preventing the occurrence of HH. Applying exogenous Ca2+ reduced the amount of apoplastic water and increased the amount of apoplastic air. We confirmed the effect of Ca2+ in reducing HH, by demonstrating that culturing the seedlings on Micro-agar medium without Ca2+ resulted in HH symptoms. This result showed that Ca2+ has a direct effect on preventing or reducing HH. Exogenously applied Ca2+ led to increases in internal ion levels of Ca2+, Cl-, and NO3 -. Correlation analysis showed Ca2+ to be significantly positively correlated with the lignin content. Moreover, we looked at the effect of Ca2+ to another component of the cell wall, namely pectin in Arabidopsis wild-type Col-0 and a less-pectin mutant (gae 6-1). Addition of Ca2+ to the medium increased total pectin content further and decreased PME activity in both genotypes. The studies of cell wall lignin, pectin and PMEs in relation with Ca2+ will help to elucidate the biochemical processes induced by the physiological state of HH. In Chapter 6, we validated the critical factors in the development of HH by investigating the roles of water retention and lignin levels. We hypothesized that capillary action might be the underlying mechanism involved in water translocation and thus might be a critical factor in development of HH. Our results showed that addition of a surfactant (Tween 20®) to the medium induced HH on Arabidopsis thaliana wild-type Col-0 in vitro seedlings. The present research showed that seedlings cultured in liquid medium led to the development of HH and an inhibition of lignin synthesis (which resulted in lower lignin content); all in line with our previous findings with seedlings grown on gelrite (Chapter 4). In order to extend further the evidence for the role of lignin on HH, we examined phenylalanine ammonia-lyase (PAL) activity and found that in hyperhydric leaves PAL activity was significantly decreased compared to normal leaves. Moreover, we observed by histochemical staining using Toluidine Blue O that the development of epidermis and cuticle on hyperhydric leaves was seriously affected in a negative way. Earlier, several individual compounds were identified that could mitigate the negative effect of culture on gelrite. Based on these results, we decided to combine three of those compounds and found that together they compensated the generally negative effect of adding CK to gelrite medium, thereby reducing the symptoms of HH in Arabidopsis seedlings. Extrapolating and validating the beneficial effect of Ca(NO3)2 in Arabidopis seedlings we studied another crop, Limonium sinuatum (Statice), and showed a reduction in HH and an increase of lignin production when applying Ca2+ to in vitro cultures of this ornamental plant. In Chapter 7, the main achievements of this study are discussed and directions for future experiments are mentioned and highlighted.
| Item Type: | Thesis (Doctoral) |
|---|---|
| Subjects: | Q Science > QK Botany |
| Depositing User: | Encik Mohd Zulkarnain Hassan bin Mohd Zainudin |
| Date Deposited: | 12 Dec 2025 11:10 |
| Last Modified: | 12 Dec 2025 11:10 |
| URI: | https://repositori.mohe.gov.my/id/eprint/137 |
Actions (login required)
 |
View Item |