Browsing by Author "Gibence, H Rose Winnie"

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Comparative expression profiling of candidate genes in the premeiotic, meiotic, and post -meiotic rice floral tissues developed in Vivo and in vitro
(Department of Molecular Biology and Biotechnology, College of Agriculture,Vellanikkara, 2025) Ammireddy Suguna; Gibence, H Rose Winnie
Rice (Oryza sativa L.), a vital staple crop, sustains the food security of over half the global population. Yet, the rising global demand for rice and shrinking arable land necessitate innovative strategies to boost productivity and resilience. Among these, investigating the genetic mechanisms underpinning reproductive processes, specifically meiosis, holds great promise for improving breeding techniques. This study focuses on analyzing the gene expression dynamics of critical candidate genes involved in meiosis across premeiotic, meiotic, and post-meiotic stages in rice florets, developed under both in vivo and in vitro conditions. Using the japonica rice cultivar Nipponbare as a model, the research aims to compare meiosis in vivo and in vitro, while assessing the feasibility of inducing meiosis in somatic cells under controlled laboratory conditions. This research employs advanced tissue culture techniques combined with molecular biology approaches to evaluate gene expression patterns and rates. Many genes like OsMEL1, OsSPO11-1, SPO11-2, OsMIL1, OsAUX1, OsMSP1, OsGAMYB1, OsARF2, OsDMC1, and OsMADS1 have been reported to play a significant role in meiosis. Key candidate genes, such as OsDMC1, OsSPO11-1, OsMADS1, OsAUX1, OsMSP1, and OsARF2, were selected for their known roles in meiotic progression and gamete formation. Media compositions were designed and optimized to mimic the microenvironment required for meiosis in pre-meiosis florets, utilizing plant hormones like auxins, cytokinins, and gibberellins. Fourteen hormonal combinations were tested to identify conditions that support meiosis induction in rice callus and pre-meiosis florets. High-quality RNA was extracted from tissues cultured under these conditions, and quantitative real-time PCR was used to profile the expression of the selected genes. Results revealed significant similarities in gene expression patterns between in vivo and in vitro systems, demonstrating the potential of in vitro models to replicate natural meiosis. Fourteen growth regulator combinations were tested using ½ MS media to support meiotic progression in rice pre-meiosis florets and callus cultures. Of these, eight combinations consistently supported growth. These included media with the following hormone combinations: 2, 4-D (2 mg/L), IBA + 6-BAP (2+1 mg/L), 6-BAP (4 mg/L), IBA (1 mg/L), 2, 4-D + IBA (2+1 mg/L), 2, 4-D (4 mg/L), 2, 4-D + 6-BAP (2+1 mg/L), and 2, 4-D + GA3(2+1 mg/L). The study aimed to support meiotic progression in vitro and evaluate the expression of six candidate genes in pre-meiosis florets. In vitro gene expression analysis revealed expected patterns for most genes in seven of the eight media, although OsMSP1 did not follow the expected trend. While expression levels varied across media, OsDMC1, OsSPO11-1, OsMADS1, OsAUX1, and OsARF2 showed significant upregulation as anticipated. For OsDMC1, the media IBA + 6-BAP (2+1 mg/L & ½ MS) showed the expected progressive fold change, with upregulation observed in both treated florets and callus compared to pre-meiotic and meiotic controls. For OsSPO11-1, the same media combination (IBA + 6-BAP) exhibited progressive fold change in treated callus, while 2, 4-D + 6-BAP (2+1 mg/L & ½ MS) also demonstrated expected results in both treated florets and callus. For OsARF2, IBA (1 mg/L & ½ MS) and 2, 4-D (4 mg/L & ½ MS) achieved the expected progressive fold change in treated florets, although callus samples did not align with expectations. For OsMADS1, 2, 4-D (2 mg/L & ½ MS) and IBA + 6-BAP (2+1 mg/L & ½ MS) showed progressive fold change in treated florets, while callus results varied. For OsAUX1, 6-BAP (4 mg/L & ½ MS) displayed a fold change similar to pre-meiotic and meiotic controls in florets, with upregulation observed in treated samples but not as expected in callus. For OsMSP1, no media demonstrated the expected progressive fold change in either treated florets or callus. Overall, the media combination IBA + 6-BAP (2+1 mg/L & ½ MS) consistently showed potential, delivering expected progressive fold change across multiple genes in both florets and callus, making it the most promising for gene expression regulation and reprogramming. The findings suggest that specific culture conditions can elicit meiosis-like gene expression patterns and potentially induce meiotic progression in somatic cells. However, further research is necessary to confirm meiotic induction and rule out aberrant cell behaviour under these conditions.
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.
Schizophyllum commune for the management of colletotrichum leaf blight of turmeric.
(Department of Plant Pathology, College of Agriculture, vellayani, 2025) Gibence, H Rose Winnie; Heera, G
The study entitled “Schizophyllum commune for the management of Colletotrichum leaf blight of turmeric’’ was carried out at College of Agriculture, Vellayani during 2021-2023, with the objective of characterization and utilization of antimicrobial compounds from S. commune for the management of Colletotrichum leaf blight of turmeric. Survey was conducted during October 2022-February 2023 in three districts of Kerala, viz., Thiruvananthapuram, Kollam and Alappuzha to study about the disease incidence (DI) and disease severity (PDI) of leaf blight of turmeric. The disease incidence ranged between 31.12 – 55.11% and disease severity between 24.32 – 62.00 %. The highest DI and PDI was observed at Neyyattinkara location of Thiruvananthapuram. The common symptom observed in surveyed area was brownish necrotic blighting from tip or margin with prominent yellow halo. Ten fungal isolates were obtained, sub-cultured and purified. Pathogenicity of these fungal isolates were proved. The pathogen was identified as Colletotrichum sp. The morphological studies of Colletotrichum sp. revealed that mycelial width, septal distance and conidial size ranged from 1.01- 1.85 µm, 13.41-7.98 µm and 9.10 × 3.70 - 18.54 × 4.50 µm2 respectively. The conidial shape was either dumbbell or oblong. Colony colour was either off white, ash grey or dark grey. Colletotrichum isolate C6 was identified as the most virulent (Sadanandapuram from Thiruvanathapuram) after virulence rating. The molecular characterization of isolate C6 was done using ITS primers where it had the maximum identity with Colletotrichum gloeosporioides. Morphological, cultural and molecular characterization is done and identified C6 isolate as C. gloeosporioides. The mycelial growth of Schizophyllum commune was either fluffy, cottony or sparse. The mycelial growth rate was maximum in SC5 (S. commune 5; DMRX-2160) followed by SC3 (S. commune 3; DMRX-2158) (1.03 and 1.01 cm day-1 respectively). SC 1 (S. commune 1; DMRX-2156), SC2 (S. commune 2; DMRX-2157), SC4 (S. commune 4; DMRX-2159) exhibited growth rate of 0.92, 0.81 and 0.69 cm day-1 respectively. Potato dextrose agar (PDA) was identified as the most suited and Czapek dox agar (CDA) least suited media for S. commune. In vitro antagonism was done by dual culture assay and poisoned food technique. All the S. commune strains were effective in managing the pathogen in vitro. The strains SC1, SC3, and SC5 exhibited lysis where as SC2 and SC4 showed overgrowth as mode of inhibition in dual culture assay. Highest inhibition was exhibited by SC5 (40.78 %) followed by SC3 (39.62%). Different concentration viz., 25, 50 and 75% of S. commune culture filtrate were tested against C. gloeosporioides in poisoned food technique. Among these, 75% concentration exhibited highest inhibition percentage. SC5 culture filtrate amended media showed highest inhibition (44.46%) of followed by SC3 (37.03%). Two most effective strains of S. commune viz., SC5 and SC3 were selected for in vivo studies. In vivo studies were conducted to evaluate effect of S. commune mycelial extract and cell free culture filtrate (CF) on leaf blight of turmeric in comparison with biocontrol agent (Pseudomonas fluorescens) and chemical control (Propiconazole). Among all the treatments spraying culture filtrate of SC5 at seven days interval showed lowest PDI (17.76%) at 14 days after inoculation (DAI) followed by propiconazole (T8), P. fluorescens (T7) and culture filtrate of SC5 (T5) which were statistically on par with each other. Similar trend was also observed at 21 and 28 DAI. Similarly, lowest lesion size observed in T5 (CF of SC5) at 14 DAI (1.96 ×1.4 cm2), 21 DAI (2.4×1.6cm2) and 28 DAI (3.96×2.3cm2). The maximum plant height was observed in T7 (127.66 cm) followed by absolute control (126.65 cm) and T5 (125 cm). There was no significant difference in the number of leaves in response to different treatments. Plant defence enzyme viz., peroxidase, polyphenol oxidase, and phenyl alanine ammonia lyase showed enhanced activity followed post inoculation the spray of P. fluorescens and culture filtrate of SC 5. Hot water extraction recovered 0.18g and 0.119g crude polysaccharide from S. commune mushroom mycelial powder and culture filtrate respectively. The results of this study revealed wide spread occurrence of Colletotrichum leaf blight in the turmeric growing areas of Kerala viz., Thiruvananthapuram, Kollam and Alappuzha. Wide variation in cultural and morphological characters of Colletotrichum isolates was observed from the surveyed areas. The most virulent isolate (C6) obtained from Sadanandapuram identified as Colletotrichum gloeosporioides based on cultural, morphological and molecular characters. PDA was identified as the most suitable media for the growth of S. commune. Schizophyllum commune strains possessed biocontrol potential with lysis and overgrowth as mode of action against C. gloeosporioides in dual culture assay. Culture filtrate of S. commune reduced the disease severity in leaf blight of turmeric. Crude polysaccharides were present in culture filtrate as well as in mushroom mycelial powder. The active compound responsible for biocontrol property of S. commune should be identified, fractionated and structurally elucidated. In vitro and in vivo activity of purified compound against fungal, bacterial and viral pathogens should be investigated and more research is needed for field level application.