PG Thesis

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    Premeiotic ovular culture and candidate gene expression studies for in vitro gamete production in rice
    (Department of Plant Biotechnology, College of Agriculture , Vellanikkara, 2024-03-20) Abhishek Gangadharan; Gibence, H Rose Winnie
    Developing genetic variation and recombining the traits of interest are critical factors affecting genetic gain in genetic engineering and crop breeding programmes. The essential steps in a breeding programme, namely, hybridisation and the repeated backcrossing and selection in the field, consume considerable time. Thus, the length of breeding cycles and the steps in breeding programmes delay the timely release of crop varieties. In vitro, gamete production can reduce the time required for this and overcome seasonal impacts and space limitations. The accumulating information on the well-mapped genetic markers and cross-over information, combined with the advanced techniques of in vitro gamete development and in vitro fertilisation, can help crop breeders recombine the desired allelic combinations in a shorter period. Meiosis involves DNA replication and two nuclear division events in germ cells. During meiotic division, DNA double-strand breaks (DSBs) are generated and are repaired via a homology-dependent DNA repair pathway (HDR), resulting in cross-over mediated genetic recombination (Mercier et al., 2015; Osman et al., 2011). During meiotic progression, the somatic cells get committed to the meiotic pathway in the S or G2 phases (Ito and Takegami, 1982). Under appropriate regulation of relevant genes, a somatic cell can behave like a germ cell to undergo meiosis and produce haploid cells, which has been achieved in the animal system. High auxin, temperature, and starvation are suggested to induce meiotic progression like changes in the cells cultured in vitro. Genes responsible for the transition from mitosis to meiosis have been elucidated in several plant species, namely, Arabidopsis, Maize, Rice, etc. OSH1, OsMADS13, OsSPL, OsAM1, OsMEL2, Os Cyclin D2, OsYUC4, OsPAIR1, and OsPAIR2 are some of the genes reported to be involved or regulated during this transition. Six genes among these viz OsMADS13, OsSPL, OsMEL2, OsCyclin D2, OsPAIR1 and OsPAIR2 were selected for analysis in the current study. Except for the OsCyclin D2, a mitosis-related gene, all others are critical to meiosis. When the expression pattern of these genes in the developing panicles of rice is analysed, one can get to know whether the panicle is undergoing premeiotic, meiotic, or postmeiotic development. Itoh and co-workers have reported that the premeiotic, meiotic, and postmeiotic panicles can be identified and isolated by following the length of the panicles (Itoh et al., 2004). In the present study, twenty-two different growth regulator combinations in ½ MS media were tested to support the growth of the post-meiotic ovules of Rice (Oryza sativa L. ssp japonica cv. Nipponbare), where seven among them were found to support the growth and development of the ovules. The seven media were with the following plant hormone combinations - IBA (1 mg/L), IBA + BAP (2+1 mg/L), 2,4-D (2 mg/L), BAP (4 mg/L), 2,4-D + IBA (2+1 mg/L), 2,4-D + BAP (2+1 mg/L) and 2,4-D (4 mg/L). One of the objectives of the current study was to support the premeiotic florets of rice to complete the meiotic progression in vitro and to monitor the expression of the six candidate genes. The candidate gene expression analysis in the premeiotic florets grown in vitro showed an expected gene expression pattern in the seven different media except for the gene OsSPL. Even though the expressions of the other genes varied between the different media conditions, OsMADS13, OsMEL2, OsPAIR1 and OsPAIR2 increased many folds in expression, as expected. The expression of OsCyclin D2 was either downregulated or remained constant under culture conditions. Further, callus culture was established from the scutellum of Oryza sativa L. ssp japonica cv. Nipponbare was subjected to the same culture conditions to test for the changes occurring at the expression levels of the candidate genes. Being cell division/meiosis-related genes, the candidate gene expression changes indicate the influence of culture conditions on the cell division mode. All the candidate genes were expressed in the callus tissues. The expression pattern of meiosis-related genes observed in ½ MS media with IBA + BAP (2+1 mg/L) was similar to the post-meiotic ovule, a sign of probable meiotic induction. However, the OsMADS13 expression level, even though upregulated, was low. All other media have shown an anomalous expression pattern for the OsSPL, OsMADS13, OsMEL2 and OsPAIR1 genes. The study shows that changing the culture conditions could induce meiosis-like gene expression patterns. When a suitable culture condition is given, it may be possible to cause and complete the meiotic transition in the somatic cells of rice. However, more studies are warranted to confirm the induction of meiosis and rule out any anomalous cell behaviour in the culture. Genes responsible for the transition from mitosis to meiosis have been elucidated in several plant species, namely, Arabidopsis, Maize, Rice, etc. OSH1, OsMADS13, OsSPL, OsAM1, OsMEL2, Os Cyclin D2, OsYUC4, OsPAIR1, and OsPAIR2 are some of the genes reported to be involved or regulated during this transition. Six genes among these viz OsMADS13, OsSPL, OsMEL2, OsCyclin D2, OsPAIR1 and OsPAIR2 were selected for analysis in the current study. Except for the OsCyclin D2, a mitosis-related gene, all others are critical to meiosis. When the expression pattern of these genes in the developing panicles of rice is analysed, one can get to know whether the panicle is undergoing premeiotic, meiotic, or postmeiotic development. Itoh and co-workers have reported that the premeiotic, meiotic, and postmeiotic panicles can be identified and isolated by following the length of the panicles (Itoh et al., 2004). In the present study, twenty-two different growth regulator combinations in ½ MS media were tested to support the growth of the post-meiotic ovules of Rice (Oryza sativa L. ssp japonica cv. Nipponbare), where seven among them were found to support the growth and development of the ovules. The seven media were with the following plant hormone combinations - IBA (1 mg/L), IBA + BAP (2+1 mg/L), 2,4-D (2 mg/L), BAP (4 mg/L), 2,4-D + IBA (2+1 mg/L), 2,4-D + BAP (2+1 mg/L) and 2,4-D (4 mg/L). One of the objectives of the current study was to support the premeiotic florets of rice to complete the meiotic progression in vitro and to monitor the expression of the six candidate genes. The candidate gene expression analysis in the premeiotic florets grown in vitro showed an expected gene expression pattern in the seven different media except for the gene OsSPL. Even though the expressions of the other genes varied between the different media conditions, OsMADS13, OsMEL2, OsPAIR1 and OsPAIR2 increased many folds in expression, as expected. The expression of OsCyclin D2 was either downregulated or remained constant under culture conditions. Further, callus culture was established from the scutellum of Oryza sativa L. ssp japonica cv. Nipponbare was subjected to the same culture conditions to test for the changes occurring at the expression levels of the candidate genes. Being cell division/meiosis-related genes, the candidate gene expression changes indicate the influence of culture conditions on the cell division mode. All the candidate genes were expressed in the callus tissues. The expression pattern of meiosis-related genes observed in ½ MS media with IBA + BAP (2+1 mg/L) was similar to the post-meiotic ovule, a sign of probable meiotic induction. However, the OsMADS13 expression level, even though upregulated, was low. All other media have shown an anomalous expression pattern for the OsSPL, OsMADS13, OsMEL2 and OsPAIR1 genes. The study shows that changing the culture conditions could induce meiosis-like gene expression patterns. When a suitable culture condition is given, it may be possible to cause and complete the meiotic transition in the somatic cells of rice. However, more studies are warranted to confirm the induction of meiosis and rule out any anomalous cell behaviour in the culture.
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    Nutritional and hormonal interventions for the improvement of yield and quality of Tomato (Solanum lycopersicum L.) under elevated CO2 and their influence on fruit shelf -life regulator (SIFSR) gene expression
    (Department of Molecular Biology and Biotechnology, College of Agriculture ,Vellayani, 2024-06-02) Susan Joshy; Manju, R V
    The study entitled “Nutritional and hormonal interventions for the improvement of yield and quality of tomato (Solanum lycopersicum L.) under elevated CO2 and their influence on Fruit Shelf-life Regulator (SlFSR) gene expression” was conducted at Depart of Plant Physiology and Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram, during 2023-2024. The objective of this study was to elucidate the effects of growth regulators and micronutrients on the yield and quality of tomato (Solanum lycopersicum L.) and on fruit shelf- life regulator (SlFSR) gene expression under elevated CO2 condition. The experiment was conducted in a completely randomized design in pot culture with three replications with tomato variety ‘Vellayani Vijay’. Plants were maintained under three conditions, open field condition (380 ppm), polyhouse (420 ppm) and Open Top Chamber (OTC) (500ppm) facility at the Department of Plant Physiology. The treatments included foliar applications of growth regulators and micronutrients T1: GA (50ppm), T2: Brassinosteroids (0.144ppm), T3: SA (50ppm), T4: B (50ppm) + Zn (50ppm) + Ca (60ppm), T5: Control, treatments were given 30 and 50 days after transplanting. Observations on growth, physiological and biochemical parameters were taken 50 days after transplanting. Yield, and quality parameters were observed at the time of harvest. The expression analysis of the SlFSR gene was done in mature fruits using Real-Time PCR. Foliar application of growth regulators like BR, GA and SA were found to improve plant growth by increasing plant height, SLA and number of branches under elevated CO2 conditions. Application of GA and Br resulted in increased chlorophyl a, and chlorophyll b contents leading to better light interception and higher accumulation of total carbohydrate. CO2 enrichment was found to increase the number of days to fruiting. Under elevated CO2 conditions of polyhouse and OTC, all the treatments including nutrient application were found to reduce the number of days taken for fruiting significantly. Earliest fruit set was recorded under T3 in polyhouse(37.01days) and under T1 in OTC (38.83days). Exposure to elevated CO2 condition, reduce the number of fruits, fruit weight and total yield. Application of GA was found to increase the yield by 12.8% under 500ppm CO2 concentration. This was due to significant increase in number of fruits produced. Plants grown under higher CO2 levels had significant reduction in quality when compared to plants grown under open condition. Quality parameters like lycopene, vitamin c and total soluble solids were increased with the application of GA, Br and SA under all the three conditions. T3 (SA) administration boosted the vitamin C content of tomato both under open and elevated CO2 conditions and recorded values of 17.61mg/g, 15.45mg/g and 13.48mg/g under open, polyhouse and OTC conditions respectively. Application of GA, SA, Br and nutrients also helped to increase Fe and P contents significantly. Application of GA and SA were found to improve pericarp thickness under ambient (7.53%) and elevated CO2 conditions (33.33%) respectively. Fruit softening and textural variations during ripening mainly result from disruptions to the cell walls of the fruit. Solanum lycopersicum L fruit shelf- life regulator (SlFSR) gene is one of the few functionally categorized GRAS genes in tomato which plays a crucial role in regulating the expression of multiple cell wall modification related genes. In the present study, SlFSR gene expression was analyzed through Real Time PCR in mature fruit tissues. Tomato fruits from plants grown under elevated CO2 showed an upregulated SlFSR gene expression. The result showed 2.8, 9.92, and 5.6- fold increase in T1, T2, and T3 respectively under eCO2 condition compared to control. In the present programme, exposure of plants to eCO2 was found to reduce shelf life of fruits significantly. All the treatments including foliar application of GA (50ppm), BR (0.144ppm), SA (50ppm), B (50ppm) + Zn (50ppm) + Ca (60ppm) at 30 and 50 DAT extended shelf life of fruits significantly in the cases of polyhouse grown plants though there was upregulation of SlFSR gene in all these cases. It is possible that other regulatory mechanisms or environmental factors could be at play delaying the onset of softening and allowing the fruit to maintain its quality for a longer period. The increase in atmospheric CO2 concentration and associated temperature build up are reported to cause tremendous reduction in fruit quality as well as shelf life in tomato. Efforts to extend shelf life to make fruits safe for long periods, keeping its original quality is of great significance The findings of the present programme revealed that foliar application of GA and SA improved yield and application of GA, Br and SA help to improve quality in terms of increased lycopene, vitamin C, Fe and P contents in frits of tomato variety Vellayani Vijay. All the treatments including foliar application of GA (50ppm), BR (0.144ppm), SA (50ppm), combined application of B (50ppm) + Zn (50ppm) + Ca (60ppm) at 30 and 50 DAT along with POP, KAU recommendations also extended shelf life of fruits significantly in the case of polyhouse grown plants. The present programme has explored the nutritional and hormonal interventions on yield, quality and the expression of the SlFSR gene under elevated CO2 conditions. The findings suggest that these interventions can significantly influence tomato physiology and biochemistry, leading to improvement in yield, fruit quality and shelf life. Future studies could further investigate effects of these interventions, focusing on molecular mechanisms, gene expression profiling and postharvest attributes. Understanding these mechanisms could pave the way for developing more sustainable and efficient agricultural practices for tomato production, ensuring food security and quality in a changing climate.
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    Effect of abiotic stress on the physiology and andrographolide content in andrographis paniculata nees.
    (Department of Plant Physiology, College of Agriculture, Vellayani, 2004) Parthasarathy, V; Roy Stephen