1. KAUTIR (Kerala Agricultural University Theses Information and Retrieval)

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Subject: search.filters.subject.Capsicum frutescens

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    Pink pigmented facultative methylotrophs (PPFMs) from chilli(capsicum frutescens) for biotic and abiotic stress migration and plant growth promotion
    (Pink pigmented facultative methylotrophs (PPFMs) from chilli(capsicum frutescens) for biotic and abiotic stress migration and plant growth promotion, 2025) Jasna Sherin, P T.; Anurajan, S
    Chilli, often referred to as the "wonder spice," is one of the most commercially valuable vegetable crop worldwide. Drought, a significant abiotic stressor, can cause substantial reductions in chilli crop yields and quality, resulting in significant economic losses. PPFMs have been shown to promote plant growth by producing plant growth regulators, altering agronomic traits, and improving water status, photosynthetic rates, and antioxidant enzyme activities. The study entitled “Pink Pigmented Facultative Methylotrophs (PPFMs) from chilli (Capsicum frutescens) for biotic and abiotic stress mitigation and plant growth promotion " was carried out during the period from 2023-2024 at College of Agriculture, Vellayani, Thiruvananthapuram, with the objective of isolation, screening and characterization of Pink Pigmented Facultative Methylotrophs (PPFMs) from chilli (Capsicum frutescens) and their evaluation for plant growth promotion with special emphasis on biotic and abiotic stress management. Thirty-five PPFM colonies were isolated from chilli phyllosphere using leaf imprint and serial dilution methods on AMS agar medium supplemented with 0.5% methanol and 10 μg cycloheximide and amended with 5% PEG 6000. These isolates were tentatively identified based on their pink pigmentation and further characterized by colony morphology, Gram reaction, and biochemical tests. Twenty-three PPFM isolates were selected for in vitro screening to assess their plant growth promoting abilities. IAA production among the isolates exhibited significant variability, ranging from 0.97 μg mL⁻¹ to 18.26 μg mL⁻¹ of culture filtrate. Isolate CF7 demonstrated the highest IAA production (18.26 μg mL⁻¹), followed by CF6 (13.60 μg mL⁻¹). All isolates produced varying amounts of gibberellic acid, ranging from 1.71 to 22.07μg mL-1. The highest gibberellic acid production was recorded in CF7 (22.07μg mL-1) followed by CF6 (21.35μg mL-1). Extracellular ammonia yield varied significantly, ranging from 0.27 to 10.06 μmol mL⁻¹. The isolate CF15 demonstrated the highest ammonia production (10.06 μmol mL⁻¹). ACC deaminase activity by different isolates ranged from 0.94 to 14.08 μg mL⁻¹, PPFM 38 (19.35 μg mL⁻¹) showed the highest activity followed by CF7 (14.08μg mL⁻¹). While all isolates grew in N-free malate bromothymol blue media, Waksman Base No. 77, and Jensen’s media, none demonstrated phosphate solubilization or antagonistic activity against phytopathogens. Based on Index ranking eight isolates were selected for primary assessment of drought tolerance potential in vitro. To evaluate drought tolerance potential in vitro, isolates were grown in media containing varying concentrations of PEG 6000. All isolates tolerated up to 30% PEG 6000. The isolates CF6, CF7, and CF15 were ranked based on drought tolerance in vitro for further studies. PCR amplification confirmed the presence of mxaF gene in these isolates, with an expected band size 550 bp indicating their ability to oxidise methanol using the methanol dehydrogenase enzyme (MDH). Molecular identification based on 16S rRNA sequencing of the isolates CF6, CF7, and CF15 showed maximum percentage identity to Methylorubrum populi, Methylorubrum thiocyanatum, and Methylorubrum podarium respectively. A pot culture experiment was conducted with chilli plants to assess the effects of PPFM isolates on plant growth promotion and drought tolerance. The experiment was a completely randomized design (CRD) with eight treatments and three replications which included four PPFM isolates (CF6, CF7, CF15, and reference culture PPFM 38), a consortium, and three control treatments (sterile water, AMS broth, and absolute control). Treatments were applied through seed treatment and foliar spray at 25th day after transplanting. The study demonstrated that application of PPFM isolates significantly enhanced plant growth parameters of chilli. The biometric parameters like maximum number of leaves (133.67), number of branches (4.50), shoot length (56.88 cm), dry weight of shoots (17.04 g), number of fruits (14.17) and yield per plant (53.45g) were recorded with CF6 treatment. Number of flowers (16), root length (35.42cm), fresh weight of roots (30.99g), and dry weight of roots (4.89g) were greater for CF15 treatment. PPFM 38 (81.83g) and CF6 (76.25g) recorded maximum fresh weight for shoots. The least parameters were recorded with either one or more of controls. Cell membrane integrity (82.3%), relative water content (85.52%), proline (86.51μg g-1tissues), SOD activity (0.44 activity g-1 min-1) and peroxidase (38.81activity g-1 min-1), were highest with PPFM 38 and CF6. The results suggest that Application of PPFMs through seed treatment and foliar spray (1%), can effectively improve growth and drought tolerance in chilli plants. Further research under field conditions is required before these can be developed as a bioformulation for sustainable agriculture.
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    Establishment of in vitro regeneration systems from callus and protoplast in capsicum frutescens L.
    (Department of Plant Biotechnology, College of Agriculture, Vellayani, 2018) Jancy J Sathyaraj; Deepa S Nair
    The present study entitled “Establishment of in vitro regeneration systems from callus and protoplast in Capsicum frutescens L. was carried out in the Department of Plant Biotechnology, College of Agriculture, Vellayani during 2015-2017. The objective of the study was to establish callus culture from different explants in C. frutescens, to establish protocol for protoplast isolation from callus/leaf mesophyll and to culture protoplast. The study was carried out in two phases viz., establishment of callus culture and organogenesis; and standardization of protocol for protoplast culture. Callus was induced from leaves and internodal segments from in vitro raised seedlings. Among the 44 treatments in MS medium with different combinations of auxins (NAA, IAA, IBA, 2,4-D and picloram) and cytokinins (BA and Kn), 100 per cent callus induction was obtained in MS media supplemented with picloram 0.50, 1.00, 1.50 and 2.00 mg L-1 (C41, C42, C43, and C44), NAA 1.50 mg L-1, NAA 2.00 mg L-1(C3, C4) and BA 3.00 mg L-1 + NAA 1.00 mg L-1 (C29). Among the 78 treatments tried for organogenesis, calli obtained from C29 treatment showed organogenesis in MS + BA 3.00 mg L-1 + IBA 1.00 mg L-1 (R37) and (MS + BA 5.00 mg L-1 + IAA 2.00 mg L-1 (R61) in 41 and 90 days, respectively. The microshoots obtained recorded 83.33 per cent rooting in MS medium supplemented with IAA 1.50 mg L-1(Rt7) in 12.83 days. Leaves excised from in vitro seedlings, and calli produced in MS + BA 3.00 mg L-1 + NAA 1.00 mg L-1 (C29), were used as explants for protoplast isolation. Leaf bits incubated in cell protoplast washing (CPW) solution containing cellulase 2.00 per cent, macerozyme 0.50 per cent and mannitol 0.50 M (maintained at pH 5.8) for 6 h (DM28) in dark at 27oC, yielded (124 x 105) protoplast per g, with a viability of 95.16 per cent. The callus yielded maximum protoplast (36 x 105 protoplasts per g) in an enzyme combination of cellulase 2.00 per cent + macerozyme 0.50 per cent + mannitol 0.60 M (maintained at pH 5.8) after 4 h 91 (DM47) of incubation under same conditions. In protoplast purification, floatation medium with 21 per cent sucrose recorded maximum protoplast yield (30 x 105 protoplasts per g tissue and 10 x 105 protoplasts per g callus) and maximum viability (90.91 per cent and 100 per cent), from leaf derived and callus derived protoplast, respectively. The purified protoplasts with 10 x105 plating density initiated microcalli in liquid MS medium supplemented with mannitol 0.50 M, 2,4- D 0.50 mg L-1 and sucrose 30g L-1 (PCM5) in 45 days. Further development to visual colony formation of microcalli was obtained on addition of liquid MS medium supplemented with mannitol 0.40 M and sucrose 5g L-1, in 60 days from callus derived protoplast and in 70 days from leaf derived protoplast. In the study, maximum callusing response was obtained in MS medium with picloram 1.50 mg L-1. Organogenesis was obtained from the calli derived in MS medium with BA 3.00 mg L-1 and NAA 1.00 mg L-1. The shoot initiated from the calli in MS medium with BA 3.00 mg L-1 and IBA 1.00 mg L-1. The rooting of microshoots could be obtained in MS medium with IAA 1.50 mg L-1. In protoplast isolation, leaf gave higher protoplast yield and viability in CPW solution with cellulase 2.00 per cent, macerozyme 0.50 per cent and mannitol 0.50 M, incubated in dark for 6 h and callus, in CPW solution with cellulase 2.00 per cent, macerozyme 0.50 per cent and mannitol 0.60 M, incubated in dark for 4 h. The protoplasts purified in 21 per cent sucrose supplemented floatation medium and adjusted to a plating density of 10 x 105, initiated microcalli in liquid MS medium supplemented with mannitol 0.50 M, 2,4- D 0.50 mg L-1 and sucrose 30g L-1. The visual colony formation of microcalli was obtained on addition of liquid MS medium supplemented with mannitol 0.40 M and sucrose 5g L-1. In this study, a callus mediated in vitro regeneration system has been established in C. frutescens. A protocol has also been developed for protoplast isolation from leaf and calli, and its culture resulting in microcalli formation.