1. KAUTIR (Kerala Agricultural University Theses Information and Retrieval)
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Item Induction of early floral meristem in saffron (Crocus sativus L.)(Department of Molecular Biology and Biotechnology, College of Agriculture,Vellayani, 2024-02-05) Aparna, S V.; Smitha BhasiThe study entitled "Induction of early floral meristem in saffron (Crocus sativus L.)" was conducted at the Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani during 2023-2024. The objective of the study was to assess the effect of various inducers in inducing early floral meristem transition, morphological analysis of different stages of bud development and differential expression of key genes involved in floral meristem transition in saffron. The experimental design followed was CRD with twenty treatments and three replications each. The explants (corms) were collected from farmer's fields in Pampore, Kashmir and subjected to a rigorous three-step surface sterilization process using 0.02% bavistin (20min), 0.04% mancozeb (20min), 1% sodium hypochlorite bleach (30min) and a final dip in 1.5% mercuric chloride solution. Surface sterilised corms were placed on Murashige and Skoog medium supplemented with various concentration of different inducers viz., GA3 (T1-T3), IAA (T4-T6), Zeatin (T7-T9), Glucose (T10-T11), Fructose (T12-T13), Sucrose (T14-T15), KNO3 (T16-T17) and Paclobutrazol (T18-T19) along with control (T20-Basal MS medium). The cultures were maintained under 24ºC temperature, 16/8hrs photoperiod with 60% humidity. 100% sprouting was noticed within 14 days of treatment using 40 mg L-1 GA3 followed by 30 mg L-1 GA3 (19 days), 10% sucrose (35 days), 5% sucrose (38 days), 10% glucose (37 days) and 5% glucose (37 days) whereas the control plants took around 45-50 days for sprouting. The bud transition from stage 1 to stage 3 was prominent in treatment using 40 mg L-1 GA3 followed by 30 mg L-1 GA3 compared to other treatments. The present study showed 40 mg L-1 GA3 as the best treatment for inducing early floral meristem in saffron. Morphological analysis focussed on the three development stages of bud based on its length viz, stage 1 (length of bud ≤ 1mm), stage 2 (length of bud between 1.5-2.0 mm) and stage 3 (length of bud ≥ 3mm) was carried out. Stage 1 was further divided into stage 1 (a) and stage 1 (b) based on morphological differences noticed around the basal region of the bud. Histological sections revealed undifferentiated floral primordium in stage 1(a) representing the vegetative phase, whereas stage 1(b) showed floral primordia initiation, representing the transition to the floral bud stage. Histological sections of stage 2 revealed prominent floral bud differentiation and initiation of perianth primordia which expanded and elongated in stage 3 accompanied by pistil primordia initiation. Molecular analysis investigated the differential expression of key genes involved in floral meristem transition, such as SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1), FLOWERING LOCUS T (FT), SEPALLATA 3 (SEP3) and CHROMATIN REMODELING 4 (CHR4). SEP3 is reported to form complexes with other floral homeotic genes to initiate differentiation of all floral organs viz sepals, petals, stamen and carpel. The differential expression analysis revealed a significant (10-fold) upregulation of SEP3 during stage 2, which correlates with the prominent floral meristem differentiation observed in stage 3, where perianth and pistil primordia became more prominent. CHROMATIN REMODELING 4 (CHR4) is a positive regulator of floral meristem transition and is also associated with the epigenetic silencing of FLC (FLOWERING LOCUS C), a crucial MADS-box transcription factor (TF) that negatively regulates floral transition. A 1.5-fold upregulation of CHR4 during the transition from stage 2 to stage 3 could be correlated with the active floral differentiation. The present investigation reveals that GA3 and sugars can be used for inducing early floral meristem in saffron. Morphological analysis confirmed that stage 1(b) marks the initiation of floral transition. Additionally, upregulation of floral meristem identity genes was observed during the floral meristem transition phase.Item Silver, magnesium and zinc nanoparticles for improving efficiency of RT-qPCR(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2022-02-10) Vinayak Mohandas; Swapna AlexThe study entitled “Silver, magnesium and zinc nanoparticles for improving the efficiency of RT-qPCR” was conducted at the Department of Plant Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram during 2019-2021. The objective of the study was to evaluate the efficacy of silver, magnesium and zinc nanoparticles in improving the efficiency of RT-qPCR. Spike samples of black pepper var. Panniyur-1 and leaf samples of rice var. Uma for nucleic acid isolation were collected from Instructional Farm, College of Agriculture, Vellayani. Efficacy of nanoparticles for improving efficiency of PCR/RT-qPCR were analyzed using housekeeping gene Actin in rice and black pepper and low copy genes OsYUCCA1 (flavin monooxygenase) in rice and TAA1 (tryptophan amino transferase) in black pepper. Primers were designed using Primer Express software for Actin1 and OsYUCCA1 in rice and reported primers were used for Actin and TAA1 in black pepper. For PCR, DNA was isolated from rice using modified Cetyltrimethylammonium bromide (CTAB) method and the good quality was confirmed using nanodrop spectrophotometer. Different concentrations (1 mgL-1 to 250 mgL-1 ) of silver (Ag), zinc oxide (ZnO) or magnesium oxide (MgO) nanoparticles (NPs) (Sigma Aldrich, USA) or their combinations were included in PCR reaction mix and PCR was performed at 95oC for 2 min followed by 35 cycles of 95oC for 15s, 48-55oC for 1 min, 72oC for 45s and final extension at 72oC for 5 min. A control was kept without any nanoparticles. Three replications were done. Efficiency and specificity of PCR were checked by comparing the intensity of the expected amplicon (213 bp) in agarose gel electrophoresis using Image lab software. Inclusion of Ag NPs and MgO NPs in PCR reaction mix at concentrations of 4 mgL-1 and 175 mgL-1 respectively, exhibited 2.3-fold and 5.7-fold increase in intensity of band. ZnO NPs at a concentration of 175 mgL-1 showed an amplification comparable to that of control (1.08-fold). Simultaneous inclusion of both Ag NPs and MgO NPs at 76 concentrations of 2 mgL-1 and 175 mgL-1 respectively in conventional PCR exhibited 6.3- fold increase in intensity of band. RNA was isolated from rice and black pepper using Trizol method and converted to cDNA. The quality of synthesized cDNA and the specificity of the primers were checked by PCR using cDNA followed by agarose gel electrophoresis. Specific amplicons of size 213 bp, 190 bp and 266 bp were obtained for Actin1 (rice), Actin (pepper) and TAA1 respectively. RT-qPCR using SYBR Green dye-based assay was carried out by including Ag (4 mgL-1 ), MgO (175 mgL-1 ) or ZnO (175 mgL-1 ) nanoparticles or their combinations in different concentrations based on the results of PCR. Technical replicates were maintained and 40 cycles of RT-qPCR was carried out at 95oC for 15s, 48-60oC for 15s and 60oC for 45s. PCR efficiency was analyzed using LinReg software. Cq values of RTqPCR were reduced in housekeeping genes and low copy number genes with inclusion of MgO nanoparticles. Among the treatments tried, MgO NPs showed maximum fold increase in amplification in all the genes analyzed (60.84, 13.70, 6.64 and 1.36-fold in Actin1, OsYUCCA1, Actin and TAA1 respectively). Combination of Ag NPs and MgO NPs exhibited 0.39-to-17.70-fold increase in amplification in RT-qPCR. Zinc oxide nanoparticles showed inhibition of amplification. To conclude, combination of Ag NPs and MgO NPs at concentrations of 2 mgL-1 and 175 mgL-1 respectively exhibited maximum improvement (6.3-fold increase) of conventional PCR efficiency. Addition of MgO NPs at a concentration of 175 mgL-1 exhibited maximum fold increase in RT-qPCR amplification (1.36 to 60.84-fold) in housekeeping and low copy number genes. Concentrations of MgO NPs and their combination with Ag NPs can be further optimized for improving PCR and RT-qPCR efficiency, especially for diagnostic detection purposes.