Browsing by Author "Smitha Bhasi"

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Biotization using piriformospora indica for induction of in vitro floral primordia in saffron(crocus sativus L.)
(Department of molecular biology and biotechnology, College of agriculture , Vellayani, 2023-09-25) Midhukrishna; Smitha Bhasi
The study entitled "Biotization using Piriformospora indica for induction of in vitro floral primordia in saffron (Crocus sativus L.)" was conducted at the Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram during 2022-23. The objective of the study was to evaluate the effect of biotization using Piriformospora indica for culture establishment and induction of in vitro floral primordia in saffron. The explants namely corms collected from farmer’s field, Jammu were used for the study. The pure cultures of P. indica were collected from the Department of Agricultural Microbiology, College of Agriculture, Vellayani. The effect of biotization of corms using P. indica was evaluated both in potting medium (Treatment 1) as well as in Murashige and Skoog (MS) medium (Treatment 2). Non-biotized corms were used as control (Treatment 3). Corms were planted in sterile potting medium (perlite and compost in 1:1 ratio) during June and kept in shade conditions for 3 months. However, no sprouting or root induction was noticed and later the corms got dried due to fungal contamination. Another strategy attempted was inducing in vitro rooting and further biotization. For this, the corms were kept at 16ºC during August-October. 100% sprouting was observed in 50 days of treatment and the sprouts thus induced in vivo were maintained at 18ºC for elongation. Further, 120-day-old elongated sprouts were cultured in basal MS medium for root induction. Healthy roots appeared in 3 days and in vitro rooted plants were transferred to potting media incorporated with P. indica mycelium. However, roots of all samples got dried in a week. For attempting biotization in vitro, full corms and 1cm3 cut corms were used for initiating sterile cultures. Sprouts were initiated within 15 days from 1cm3 cut corms cultured in MS medium supplemented with 1.5ppm BAP (6 benzylaminopurine). Further, the sprouts induced both in vitro and in vivo were subjected for elongation in basal MS medium. Significant increase in shoot length was noticed in sprouts induced in vivo (kept at 16ºC) compared to sprouts induced in vitro. Flower buds were induced from elongated sprouts by sub culturing in MS 81 medium and 50% sprouts that were induced in vivo, developed in vitro floral buds. However, no floral buds were observed in elongated sprouts induced in vitro. Biotization of in vitro cultures (Treatment 2) was carried out using P. indica agar discs and derivatives of P. indica. 130-day-old elongated sprouts having in vitro roots were inoculated with P. indica agar discs in MS medium for colonization. However, colonization was not observed under microscopic analysis. 165-day-old elongated sprouts were also treated with derivatives of P. indica and compared with control. Elongated sprouts treated with derivatives of P. indica exhibited a significant increase in plant height compared to control. These sprouts were further sub-cultured for a period of 7 weeks in MS medium for inducing multiple shoots. 165-day-old elongated plantlets treated for 80 days with derivatives of P. indica produced more number of multiple shoots per corm compared to control. Further, the multiple shoots developed were cultured in MS medium supplemented with 9% sucrose for inducing in vitro cormlets. Multiple shoots pre-treated with P. indica produced larger and more number of cormlets compared to control. To conclude, sterile potting media (perlite-compost) was found not suitable for the establishment and growth of corms under field conditions of Vellayani region. Storage of corms at 16ºC during dormant stage is found critical for in vitro growth, rooting and flowering in saffron. In vitro treatment using derivatives of P. indica in saffron showed positive responses with respect to plant height, number of multiple shoots, number, and size of in vitro cormlets. Hence, derivatives of P. indica can be used for better in vitro establishment of cultures, multiple shooting, and in vitro cormlet production in saffron.
In-vitro chemotherapy for inducing tolerance towards Tomato leaf curl New Delhi virus (ToLCNDV)in bitter gourd (Momordica charanita L.)
(Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani, 2023-12-12) Adarsh, M V.; Smitha Bhasi
The study entitled “In-vitro chemotherapy for inducing tolerance towards Tomato Leaf Curl New Delhi virus in bitter gourd (Momordica charantia L.)” was conducted at the Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram during 2022-23. The objective of the study was the evaluation of in-vitro treatment with antiviral compounds, viz., Ribavirin, Virus–Ex and extracts of Bougainvillea spectabilis for inducing tolerance towards Begomovirus- ToLCNDV in bitter gourd (Momordica charantia L.). The explants namely seed of bitter gourd variety Preethi was collected from, Instructional farm, College of agriculture, Vellayani. Seeds were subjected to hot water treatment at 55°C for 15 minutes, followed by washing in 0.2% bavistin for 15 minutes and kept in dark for inducing germination. 91.2% germination was noticed in 3 days. The germinated seeds were grown in Murashige and Skoog (MS) medium supplemented with 2ppm BA and 20ppm concentration of various antiviral compounds viz., Ribavirin (Treatment 1), Virus-Ex (Treatment 2), extract of Bougainvillea spectabilis (Treatment 3) along with control. Percentage of culture establishment was found to be maximum (65.25%) in ribavirin treated plants followed by virus ex and bougainvillea. 20 days old in-vitro grown plants were hardened in coir pith compost and the biometric observations were taken and compared. Significant increase in plant height and number of leaves over control was noticed in treatment using ribavirin and virus ex respectively. DNA was isolated from 20 day old (hardened) treated and control plants prior to whitefly transmission and were confirmed to be virus free using PCR. Whitefly mediated artificial inoculation of virus in hardened plants (25 day old) was done and the plants were maintained inside insect proof cage for symptom development. Symptoms were observed in control plants after 10 days of whitefly transmission. Further PCR was performed to confirm the presence/absence of Begomovirus. Absence of virus in treatments with ribavirin and virus ex were 59 confirmed in PCR. Treatment using extracts of bougainvillea did not show any symptoms but confirmed positive in molecular detection. The control plants which developed symptoms was found positive in PCR. Peroxidase assay (Lobenstein and Linsey method) showed maximum increase in activity (3-fold) in plants treated with ribavirin followed by extracts of bougainvillea (2-fold) three days after whitefly transmission. Biometric observations on 35th day viz., plant height, number of leaves and leaf area were found higher in all the treatments compared to control, out of which treatment using ribavirin was found to be highly significant for all the parameters. To conclude, the present study in bitter gourd could confirm the antiviral activity of ribavirin and virus ex towards Begomovirus through whitefly mediated transmission test and molecular detection using PCR
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 Bhasi
The 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.
Isolation, characterisation and evaluation of PINI and BP genes in ralation to inflorescence architecture in black pepper (Piper nigrum L.)
(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2019) Smitha Bhasi; Swapna Alex
Morphomolecular charecterisation of the variants of piper nigrum L. variety panniyur -1
(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2008) Smitha Bhasi; Swapna Alex
The study entitled “Morphomolecular characterization of variants of Piper nigrum L. variety Panniyur-1” was conducted at the Department of Plant Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram and in the Block V of Panniyur-1 at the Regional Agricultural Research Station (RARS), Ambalavayal during the year 2006-2007 with an objective of characterizing the variants of black pepper variety Panniyur-1 based on morphological traits and RAPD profiles. Black pepper often referred to as the ‘King of spices’ is the most important spice in the world. The first ever hybrid of black pepper, Panniyur-1 (Uthirankotta x Cheriyakaniyakadan) is the most popular pepper variety grown in India and also in Kerala. In black pepper, propagation through cuttings is being practiced for decades for producing true-to type plants. However, contrary to this belief, there are reports for the existence of variability. Variability was reported even at the intraclonal level. The first such report in black pepper was in the local variety Karimunda (Ratnambal et al., 1985). According to Pradeepkumar et al. (1999), there exists intra-clonal variability in yield among the hybrid clone Panniyur-1 at the RARS, Ambalavayal. Such reports deserve serious concern and in depth analysis as pepper is a leading commercial crop of India, important in the domestic as well as international markets. The present study was taken up in this context utilising the progeny of the forty variant plants reported by Pradeepkumar et al. (2003) from the RARS, Ambalavayal. The objective was to assess the extent of variability with respect to morphological traits including yield parameters as well as the molecular analysis of genetic variability. On morphological analysis of the forty plants, considerable variation was observed. The maximum variation was observed in number of berries per spike followed by drying percentage. The analysis of the dendrogram showed that none of the plants were 100 per cent similar at a distance of 1.0. At a distance of 2.0 the clones can be grouped into five clusters. At a distance of 10, the plants can be grouped into two clusters comprising a major group with twenty-nine plants and a minor group with eleven plants. Molecular analysis also revealed variability, accounting for 66.34 per cent polymorphism. In the dendrogram at the similarity index 0.70 the plants grouped into two major clusters indicating thirty per cent dissimilarity. None of the plants were showed 100 per cent similarity. All the forty plants under study formed individual clusters at a similarity index 0.91 except V36 and V37. Ninety percent similarity was observed between the plants V20 and V30. At a similarity index below 0.70 the dendrogram showed a cluster including all the plants except V14. The present findings need further confirmation with more number of primers and other molecular markers like ISSR, AFLP etc. The occurrence of variability among the clones of Panniyur-1 in other major pepper growing tracts also needs to be investigated in detail.
Plant growth regulators for enhancing in vitro cormlet production in saffron(crocus sativus)
(Department of Molecular Biology and Biotechnology, College of Agriculture,Vellayani, 2024-07-22) Neethu, R S.; Smitha Bhasi
The study entitled “Plant growth regulators for enhancing in vitro cormlet production 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 evaluate the effect of plant growth regulators in enhancing in vitro cormlet formation and differential expression of key genes involved in cormlet production. Experimental design followed was CRD with five treatments and three replications each. The explant, 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 solutions (30min) and a final dip in 1.5% mercuric chloride solution. In vitro multiple shooting attempted using Murashige and Skoog (MS) medium supplemented with 2ppm Benzyl adenine (BA) showed sprouting, but did not grow further. Corms maintained under in vivo conditions of 16ºC temperature, 16/8hrs photoperiod and 60% humidity showed 100 per cent sprouting in 65 days. The sprouted corms were transferred to basal MS medium for elongation and shooting. Healthy multiple shoots were observed in 15 days with an average of 5 multiple shoots per culture and profuse roots were noticed in 2 days in all replications. Further, the in vitro multiple shoots were transferred to half-strength MS medium supplemented with 20 μM thidiazuron (TDZ), 10 μM indole acetic acid (IAA), 40% sucrose, and 0.25% of various anti-gibberellins viz., chlorocholine chloride (CCC), paclobutrazol (PBZ), chlormequat chloride (CMC) and mepiquat chloride (MPQ) along with control. Cultures maintained in full strength MS medium was used as absolute control. Cormlet initiation was first noticed in treatment using MPQ on 10th day followed by CCC (20 days), control (20 days) and absolute control (30 days). Cormlets were initiated in all 3 replications within 16 days in treatment using MPQ followed by CCC (36 days) and control (38 days). Total number of cormlets obtained was highest in treatment using MPQ (7 nos), followed by CCC (5 nos), control 64 (5nos), CMC (4nos) and absolute control (1no). The present study showed 0.25% MPQ as the best treatment with early initiation and maximum number of cormlet production. In vitro cormlets generated in treatment using 0.25% MPQ showed prominent size compared to control. In the treatment using 0.25% mepiquat chloride significant reduction was noticed in the expression level of GA3OX (0.08-fold) and upregulation of all the key genes involved in starch biosynthesis viz., SS (2.3-fold), SUSY (1.3-fold), PGM (1.1 fold). Similar results were noticed in treatment using 0.25% CCC, whereby significant reduction in the expression level of GA3OX (0.03-fold) and upregulation of SS (1.3-fold) and SUSY (1.8-fold) was found. Molecular studies could reveal a negative co-relation in the expression pattern of genes involved in biosynthesis pathway of gibberellin and starch metabolism during the early phase of cormlet initiation. To conclude, plant growth regulators viz., mepiquat chloride and chlorocholine chloride can be used effectively for early cormlet initiation and to enhance in vitro cormlet production in saffron. The inhibitory activity of MPQ & CCC against GA3oxidase noticed in the present study suggest the potential of these compounds in blocking the conversion of inactive GA to bioactive GA. The present study is the first report on differential expression analysis of key genes involved in in vitro cormlet formation in saffron and could reveal a low gibberellin mediated starch accumulation during early phase of cormlet production.