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Kerala Agricultural University, Thrissur

The history of agricultural education in Kerala can be traced back to the year 1896 when a scheme was evolved in the erstwhile Travancore State to train a few young men in scientific agriculture at the Demonstration Farm, Karamana, Thiruvananthapuram, presently, the Cropping Systems Research Centre under Kerala Agricultural University. Agriculture was introduced as an optional subject in the middle school classes in the State in 1922 when an Agricultural Middle School was started at Aluva, Ernakulam District. The popularity and usefulness of this school led to the starting of similar institutions at Kottarakkara and Konni in 1928 and 1931 respectively. Agriculture was later introduced as an optional subject for Intermediate Course in 1953. In 1955, the erstwhile Government of Travancore-Cochin started the Agricultural College and Research Institute at Vellayani, Thiruvananthapuram and the College of Veterinary and Animal Sciences at Mannuthy, Thrissur for imparting higher education in agricultural and veterinary sciences, respectively. These institutions were brought under the direct administrative control of the Department of Agriculture and the Department of Animal Husbandry, respectively. With the formation of Kerala State in 1956, these two colleges were affiliated to the University of Kerala. The post-graduate programmes leading to M.Sc. (Ag), M.V.Sc. and Ph.D. degrees were started in 1961, 1962 and 1965 respectively. On the recommendation of the Second National Education Commission (1964-66) headed by Dr. D.S. Kothari, the then Chairman of the University Grants Commission, one Agricultural University in each State was established. The State Agricultural Universities (SAUs) were established in India as an integral part of the National Agricultural Research System to give the much needed impetus to Agriculture Education and Research in the Country. As a result the Kerala Agricultural University (KAU) was established on 24th February 1971 by virtue of the Act 33 of 1971 and started functioning on 1st February 1972. The Kerala Agricultural University is the 15th in the series of the SAUs. In accordance with the provisions of KAU Act of 1971, the Agricultural College and Research Institute at Vellayani, and the College of Veterinary and Animal Sciences, Mannuthy, were brought under the Kerala Agricultural University. In addition, twenty one agricultural and animal husbandry research stations were also transferred to the KAU for taking up research and extension programmes on various crops, animals, birds, etc. During 2011, Kerala Agricultural University was trifurcated into Kerala Veterinary and Animal Sciences University (KVASU), Kerala University of Fisheries and Ocean Studies (KUFOS) and Kerala Agricultural University (KAU). Now the University has seven colleges (four Agriculture, one Agricultural Engineering, one Forestry, one Co-operation Banking & Management), six RARSs, seven KVKs, 15 Research Stations and 16 Research and Extension Units under the faculties of Agriculture, Agricultural Engineering and Forestry. In addition, one Academy on Climate Change Adaptation and one Institute of Agricultural Technology offering M.Sc. (Integrated) Climate Change Adaptation and Diploma in Agricultural Sciences respectively are also functioning in Kerala Agricultural University.

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2004 - 2009492010 - 2016102
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Subject: Plant Biotechnology × End date: 2016 × Type: Thesis ×
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Now showing 1 - 9 of 151
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    ThesisItemOpen Access
    Cryopreservation of chethikoduveli (Plumbago rosea L.) and assessment of genetic fidelity of regenerated plantlets using molecular markers
    (Department of Plant Biotechnology, College of Agriculture, Vellayani, 2014) Anand, Vishnu Prakash; KAU
    Investigations on “Cryopreservation of Chethikoduveli (Plumbago rosea L.) and assessment of genetic fidelity of regenerated plantlets using molecular markers” were carried out at the Department of Plant Biotechnology, College of Agriculture, Vellayani during 2011-2013. Plumbago rosea var. Agni plants were collected from AMPRS, Odakkali, Ernakulam and maintained at the Department of Plant Biotechnology, College of Agriculture, Vellayani as source of explant during the course of the study. The objectives of the present study was to standardise cryopreservation protocol by encapsulation dehydration technique for long term conservation of P. rosea and genetic fidelity assessment of plantlets recovered and regenerated from cryostorage using molecular markers. The project was carried out in two phases viz., in vitro regeneration and in vitro conservation by cryopreservation of P. rosea. In vitro regeneration protocol was optimised for P. rosea var. Agni. Various steps of in vitro regeneration viz., surface sterilization, axillary shoot proliferation, in vitro rooting and acclimatization and planting out has been standardised. For surface sterilizing, single nodal explants (3-4 cm long) were subjected to fungicide treatment with 0.1 per cent carbendazim 50 per cent W. P. (for 30 min) followed by aseptic sterilisation dip with absolute alcohol. Further, the explants were surface sterilised with 0.2 per cent mercuric chloride (for 5 min) which gave 100 per cent survival without any contamination. Enhanced release of axillary buds from single nodal explants, with maximum shoot proliferation (5.28 shoots/culture) was obtained in the medium, MS + BA 1.5 mg l-1 + IAA 1.0 mg l-1. The best response (10.67 roots/culture) of in vitro rooting of plantlets was obtained in the medium, MS + NAA 1.0 mg l-1. In vitro rooted plants gave a maximum survival rate of 76 per cent and 72 per cent, when planted out in potting media consisting of red soil and coir pith (3:1) and red soil and coir pith (2:1) supplemented with VAM respectively at 25 per cent shade. In cryopreservation studies, preconditioning treatment (sucrose 0.5 M for 7 days) recorded maximum shoot proliferation (2.67 shoots/culture) when nodal segments with single axillary bud were cultured on MS + BA 1.5 mg l-1 + IAA 1.0 mg l-1 medium. Among different encapsulation treatments, maximum shoot proliferation of (2.31 shoots/culture) was obtained in beads formed with sodium alginate 2.5 per cent and calcium chloride 100 mM, when cultured on the medium, MS + BA 1.5 mg l-1 + IAA 1.0 mg l-1. Pre-culture medium supplemented with sucrose 0.5 M for 3days gave maximum shoot proliferation (3.44 shoots/culture) when cultured on the medium, MS + BA 1.5 mg l-1 + IAA 1.0 mg l-1. A desiccation duration of 5 h at 18.13 per cent moisture level was found to be most effective giving 66.67 per cent survival and 62.50 per cent regeneration on thawing and culturing on the recovery medium MS + BA 1.5 mg l-1 + IAA 1.0 mg l-1. The beads when stored in liquid nitrogen for different duration and cultured on recovery medium did not show any significant variation with respect to survival per cent. RAPD markers were tried to study the genetic fidelity of the regenerated plantlets from encapsulated and cryopreserved axillary buds. Six primers were screened and RAPD banding patterns of the cryoregenerated plantlets and control plants were compared. Polymorphism was not found with any of the primers tested. RAPD profiles of cryoregenerated plantlets were identical to those of the control. The in vitro regeneration protocol optimized included surface sterilization of single node cuttings with 0.2 per cent HgCl2 for 5 min, axillary shoot proliferation in MS medium supplemented with BA 1.5 mg l-1 and IAA 1.0 mg l-1, in vitro rooting in MS medium supplemented with NAA 1.0 mg l-1 and planting out in potting medium, red soil and coir pith (3:1). The protocol for encapsulation dehydration technique of cryopreservation was standardised for the axillary buds of P. rosea with preconditioning in semi solid MS medium supplemented with sucrose 0.5 M for 7 days, encapsulation using sodium alginate 2.5 per cent and calcium chloride 100 mM followed by pre-culture in liquid MS supplemented with sucrose 0.5 M for 3 days and 5 h dehydration (MC 18.13 %), rapid freezing in LN for at least 2 h and recovery in the medium MS + BA 1.5 mg l-1 + IAA 1.0 mg l-1. The cryopreservation protocol using encapsulation-dehydration technique standardised could be utilised for long-term conservation of P. rosea.
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    ThesisItemOpen Access
    Development of an in vitro regeneration system and validation of genetic stability in phalaenopsis hybrid winter spot with molecular marker
    (Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2016) Asha Amal, Raj; KAU; Lissamma, Joseph
    Phalaenopsis “Moth Orchids” are among the most beautiful flowers in the world. This genus has economic value for pot plant and cut flower production and is distributed throughout Southeast Asia. Most popular method of propagation for orchid is through in vitro propagation, as it produces large number of clones in relatively short duration. Despite its potential to produce numerous plants from a single leaf segment, it is liable to unpredictable mutations or somaclonal variation during the process of multiplication. Variation can arise due to many reasons such as type of media, plant growth regulators and its concentration, type of explants and number of subculture cycles. The percentage of the variation can range from 0-100% depending on varieties with an average of 10% among Phalaenopsis (Tokuhara and Mii, 1993). So the present investigation on “Development of an in vitro regeneration system and validation of genetic stability in Phalaenopsis hybrid Winter Spot with molecular marker” was taken up at the Center for Plant Biotechology and Molecular Biology, College of Horticulture from 2013-2016. Flowering mother plants of Phalaenopsis hybrid Winter Spot were used as explant source. Among the explants namely inflorescence node, transverse thin cell layer of leaf and root segments used for tissue culture study in this orchid, inflorescence node was the best with respect to culture response. The best surface sterilization treatment for leaf explants identified was treatment 0.1% bavistin + prill 2 drops (30 min) and 0.1 per cent HgCl 2 ( 8 min) which give maximum per cent of culture survival and minimum contamination rate. The best surface sterilization treatment for inflorescence node identified was treatment with 0.1% bavistin + 2 drops prill (30 min) , one minute dip in 70 per cent ethanol and 0.1% HgCl 2 (7 min).From different basal media (full MS and 1⁄2 MS) tried, response was observed only in the medium of Full MS for inflorescence node. Among the different growth regulators tried, MS medium supplemented with BA and TDZ was found to give good shoot regeneration from inflorescence node explants. MS +2mgl -1 TDZ recorded highest percentage (80%) of culture establishment, followed by MS + 4.5 mgl -1 of BA (55%) per cent of sprouting. Among the explants tried, only inflorescence node responded with sprouting. Root segment remained as such without any change, whereas leaf explants remained green up to 2 weeks, thereafter started drying in all the growth regulators combination. For induction of multiple shoot, MS medium supplemented with 4.5 mgl -1 BA resulted in the highest average number of multiple shoot (4.15). Elongation and rooting was observed in MS medium supplemented with BA 4.5mgl-1 +IAA 1mgl -1 with 80 % rooting. Root initials were observed 50 days after inoculation. The potting media, charcoal, brick pieces and sphagnum moss in the ratio of 1:1:1 was found ideal for hardening of Phalaenopsis hybrid winter spot with 100% survival. Genetic stability studies using RAPD marker were carried out with the mother plants along with three regenerants each. Six primers were selected based on DNA amplification pattern. In RAPD assay, M1 mother plant recorded the highest average polymorphism of 19.7% and M3 mother plant recorded the least average polymorphism of 8.18%. Using NTSYS software, the similarity coefficients for first, second and third plant between M1 mother plant, M2 mother plant and M3 mother plant and corresponding regenerants were 0.91, 0.92 and 0.93 respectively. In fourth plant, the similarity coefficient exhibited 100% similarity between mother plant, the first clone C1 and third clone C3. The established micropropagation protocol can be used with suitable modification for large scale production of other Phalaenopsis varieties.
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    ThesisItemOpen Access
    Isolation and Characterization of cDNA encoding chalcone synthase from flower buds of orchid Dendrobium variety sonia 17
    (Department of Plant Biotechnology, College of Agriculture, Vellayani, 2007) Anjana, G R; KAU; Soni, K B
    The study entitled “Isolation and characterization of cDNA encoding chalcone synthase gene from the flower buds of orchid Dendrobium variety Sonia 17” was conducted at the Department of Plant Biotechnology, Vellayani, Thiruvananthapuram during the period from 2005 to 2007 with an objective of studying the isolation and characterization of cDNA encoding chalcone synthase gene involved in anthocyanin pigmentation in orchid flower buds. . Heterologous forward and reverse primers were designed based on the gene sequences of Oryza sativa, Fragaria ananasa and Phalaneopsis orchid using primer3 software. Total RNA was isolated from immature floral tissues using hot phenol method which gave an yield of 80 - 200 μg g -1 of the tissue and a A260/A280 ratio ranging between 1.6 –2.0. Messenger RNA was purified from the total RNA using the mRNA purification kit from GENEI (Bangalore). Reverse transcription-polymerase chain reaction was carried out to study the expression of gene. The RT-PCR amplified products representing chalcone synthase (CHS) gene was eluted and purified. The product was sequenced and studied the similarity of the same using homology search. All sequenced regions were subjected to BLASTN and BLASTX similarity search. Rice chalcone synthase specific primer produced an amplified sequence of 460 bp long and showed maximum similarity to the cDNA clone 5', mRNA sequence of. flower bud of Phalaenopsis violacea and flower bud of Phalaenopsis equestris Lambda ZapII cDNA Library in BLASTN similarity search.BLASTX analysis of the sequence showed similarity to maturase K protein of Aerangis kirki. The cDNA amplified with strawberry chs specific primer showed maximum similarity to the cDNA clone 5’, mRNA sequence of Phalaenopsis violacea flower bud and flower bud of Phalaenopsis equestris in the BLASTN similarity search. BLASTX analysis of the sequence showed similarity to LFY-like protein of Serapias lingua. The results of the nucleotide to nucleotide search (BLASTN) of the cDNA of orchid, amplified using chalcone synthase specific primer from orchid showed similarity to cDNA 5', mRNA sequence of Ipomoea batatas in the BLASTN similarity search. BLASTX analysis of the sequence showed similarity to retrotransposon protein of Oryza sativa (japonica cultivar-group). The result of the sequences obtained from the study shows similarity with the genes involved in the biosynthetic pathway of Phalaenopsis orchid flower fragrance.
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    ThesisItemOpen Access
    Management of biodegradable plant tissue culture lab wastes through biomethanogenesis
    (Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2008) Abdulla Fayas, T; KAU; Rajendran, P C
    Generating renewable source of energy from tissue culture laboratory waste by the process of biomethanogenesis is the focal theme of present investigation. Unlike developed countries, the developing countries are hesitant to establish more number of biotechnology/ tissue culture laboratories due to financial constraints. Easy and regular availability of biogas from TC wastes will be a boon to establish self-sustainable TC laboratory in view of present energy The biogas experimental units required for the study was designed and various treatments were employed for the biodegradation of tissue culture waste, using the methanogenic bacteria Methanobacterium ruminatium, Methanobacterium formicicum; Methanosarcina barkeri, Bactereoides ruminicola, Selenomonas ruminatium, Eubacterium tortuosum and Clostridium butyricum. Treatment involving TC waste and cow dung was also conducted for biomethanation in the present study. Quantity of gas production and its combustibility was noticed for various treatments. In bacterial treatments the quantity of gas generation was highest for Clostridium butyricum. Only treatments involving cow dung produced combustible gas. Molecular characterization, of methanogenic bacterial cultures was also done for finding the genetic similarity between them. RAPD followed by scoring . of the bands by UPGA routine showed maximum similarity between bacterial cultures of Methanobacterium ruminatium and Methanobacterium jormicicum with Methanosarcina barkeri. Physio-chemical characters like CIN ratio of the TC wastes, pH and temperature of medium and Hydraulic retention time was also observed for the various treatments. The CIN ratio of the TC wastes was found to be very low and nowhere near the optimum CIN ratio of 20-30 required for gas production. Other parameters like pH of the treatments and Hydraulic retention time was also. • noticed. The pH of the treatments involving bacterial cultures was very low, considering the normal pH of 6.8 to 7.5 required in biogas generation. The main constraints in the biogas generation were found out to be the low CIN ratio of the TC waste and the low pH of the medium. The present study indicated the possibility ofbio-gas generation from TC waste through fortification using various supplements like coconut water and coir pith which have higher CIN ratio.
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    ThesisItemOpen Access
    Characterisation of Pathogenesis related proteins for anthracnose resistance in vegetable cowpea, Vigna spp.
    (Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2013) Agatha Shiny, A; KAU; Deepu Mathew
    Cowpea (Family: Fabaceae) is an important pulse cum vegetable crop of suitable for the tropical and sub-tropical regions of the world. The grain type cowpeas better tolerates the biotic and abiotic stresses against the vegetable types. Under humid conditions, vegetable types, especially the pole types are susceptible to many diseases and among them, anthracnose caused by Colletotrichum lindemuthianum (Sacc. & Magn.) Br. and Cav. is very severe. In Kerala, complete yield loss in vegetable cowpea is reported due to anthracnose during monsoons. The study entitled “Characterization of pathogenesis related proteins for anthracnose resistance in vegetable cowpea, Vigna spp.” was carried out with objective to develop the protein profiles of resistant and susceptible bush and pole genotypes through SDS-PAGE analysis at different time intervals of infection and to characterize the differentially expressed proteins by MALDI-TOF followed by in-silico analyses. Two bush type varieties Pusa Komal and Kanakamony, the former reported to be highly susceptible and the latter immune to anthracnose and two pole type varieties Lola and Arimbra Local, of which the former susceptible and the latter resistant were used in the study. Pure culture of the pathogenic fungus was developed and maintained on selective medium (Neopeptone-Glucose-Agar) at the Dept. of Plant Pathology. The identity of Colletotrichum lindemuthianum has been established from the spore characteristics observed under phase contrast microscope and the pathogenicity was confirmed through artificial inoculation under controlled conditions. The pot culture experiment was conducted with 50 pots per variety. Artificial inoculation of pathogenic fungus was done and the leaf samples were collected at 0, 6, 12, 18, 24, 48, 72, 96, 120, 144,168 and 192 hours after artificial inoculation. The total protein was extracted using Tris-HCl buffer (pH-7.5), quantified using spectrophotometer and analyzed by SDS-PAGE method. The defense enzymes like peroxidase (PO), polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL) were assayed. By artificial inoculation, disease responses for anthracnose were confirmed to be highly susceptible in Pusa Komal and Lola; highly resistant in Arimbra Local and immune in Kanakamony. Protein expression was found to be higher from the initial hours in resistant varieties whereas in susceptible varieties, the expression was reduced immediately after infection then peaked at 18hr and gradually decreased later on. Two prominent and differentially expressed protein bands at 56 kD and 14 kD were sequenced in MALDI-TOF to obtain the peptide mass fingerprint. Through in-silico analyses using Mascot server software, they were identified to be the large and small subunits of the chloroplastic enzyme RuBisCo. Thus the capability of a variety to maintain high levels of RuBisCo was found to be the deciding factor for anthracnose disease resistance. Further, protein profiles developed after purification of proteins by dialysis have clearly identified the differentially expressed band at 29 kD in the resistant varieties which is in the size range of already reported PR proteins. PO and PAL activities were proportionate to the resistance behavior, with the peak values at 18 and 24 hr after inoculation. With the results of this study, these defense enzymes are recommended as biochemical markers for identifying the resistance in the accessions. Capability to maintain higher levels of RuBisCo, PO and PAL enzymes is the characteristic of anthracnose resistant vegetable cowpeas and the future breeding programmes could be oriented in this direction
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    ThesisItemOpen Access
    DNA fingerprinting of selected black pepper (piper nigrum L.) varieties.
    (Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2011) Manjunath, Mogalayi; KAU; Nazeem, P A
    Black pepper (Piper nigrum L.) famous as “Black Gold” and also known as “King of Spices” is one of the important agricultural commodities of commerce and trade in India since pre-historic period. The crop is the major source of income and employment for rural households in the predominantly pepper growing State of Kerala where more than 2.5 lakh farm families are involved in pepper cultivation. Karnataka, Tamil Nadu are the other major pepper producing States in the country. Kerala accounts for 80-90% of the total pepper production in the country. Idukki and Wynadu are the two major pepper producing districts in Kerala. Different cultivars/varieties are popular among the farmers and there phenotypic identity is not very distinct. The study entitled “DNA fingerprinting of selected black pepper (Piper nigrum L.) varieties” was carried out at the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture during the period 2009-2011. The objectives of the study were to characterize the released black pepper varieties of KAU using different molecular markers - RAPD, ISSR and SSR and to develop DNA fingerprint with which the variety could be identified and its fidelity detected. Seven black pepper varieties (Panniyur 1 to Panniyur 7) collected from Pepper Research Station, Panniyur and maintained at CPBMB, COH were used for the study. DNA extraction was done with CTAB (Rogers and Benedich, 1994) method with slight modification. The RNA contamination was completely removed through RNase treatment. Good quality DNA with UV absorbance ratio (A260/A280) 1.80 - 1.89 was used for further analysis. The PCR conditions were optimized for RAPD, ISSR and SSR assay. 30 RAPD, 34 ISSR and 29 SSR primers were screened with bulked DNA of black pepper varieties for amplification and those which gave reliable distinct banding pattern were selected for further amplification and fingerprinting. The PCR products obtained from RAPD, ISSR and SSR analysis were separated on 1.3 to 2 percent agarose gel and the amplification patterns recorded. The genomic DNA from each variety was amplified with 10 each of selected RAPD and ISSR primers and 8 SSR primer pairs. The amplification pattern was scored and depicted to develop fingerprint for each variety. The Resolving power (Rp) worked out for the different primers ranged between 7.42 to 9.42 in RAPD and 5.42 to 12.28 in ISSR analysis; indicating the capacity of the primers selected to distinguish the varieties. The Polymorphic Information Content (PIC) varied from 0.86 to 0.90 for RAPD analysis and it was between 0.80 and 0.89 in ISSR analysis indicating the variability among the genotypes. Distinct bands were used to develop DNA fingerprint of black pepper varieties Panniyur 1 to Panniyur 7 through RAPD, ISSR and SSR analysis. Sharing of amplicons developed for each primer with other varieties was also analyzed and demarcated with different colour codes in the fingerprints developed. Most of the amplicons were found shared among the varieties. However, the pattern of sharing was different and good enough to separate out the varieties. Combined DNA fingerprint of each variety with RAPD, ISSR and SSR data was also developed. The amplification pattern observed in RAPD, ISSR and SSR analysis was scored and analyzed for quantifying the variability among the varieties. The computer package NTSYS-Pc was used for cluster analysis. Maximum variability observed was 48 percent for the variety Panniyur 4. The varieties Panniyur 1 and Panniyur 3 having the same parentage indicated 76 percent similarity. The fingerprint developed was good enough to provide varietal identity and the analysis could reveal variability/relatedness among the seven varieties. Separate and combined fingerprints were developed for all the seven varieties through RAPD, ISSR and SSR analysis. The DNA fingerprints thus developed could be utilized for the variety registration and settling IPR issues.
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    ThesisItemOpen Access
    In vitro shoot regeneration and micrografting in nutmeg (Myristice fragrans houtt.)
    (Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2008) Liffey Zachariah, Antony; KAU; Valsala, P A
    Nutmeg (Myristica fragrans Houtt.), is dioecious and dimorphic in branching habit with erect growing orthotropic and horizontally growing plagiotropic shoots. The long gestation period and dioecious nature of the crop causes difficulty in the production of quality planting materials of known sex. Vegetative propagation, budding and grafting with orthotropic scion material produces erect growing tree with upright tree architecture. In vegetative propagation, scarcity of orthotropic scion material is a limiting factor in large scale production of planting materials. So the programme “In vitro shoot regeneration and micrografting in nutmeg (Myristica fragrans Houtt.)” was taken up. The objectives of the study were: (1) To identify the culture conditions for multiple shoot production from orthotrops of gynoecious plants of nutmeg through enhanced release of axillary buds and shoot tip culture and (2) To standardize micrografting technique with in vitro and in vivo shoots as scion. The work was done at CPBMB, College of Horticulture, Vellanikkara. SH medium (Schenk and Hildebrandt, 1972) was found to be the best basal medium for in vitro culture establishment of nodal segments of nutmeg compared to ½ MS (Murashige and Skoog, 1962) and WPM (Lloyd and Mc Cown, 1980). Surface sterilization of nodal segments by soaking in (0.1%) carbendazim for 10 minutes followed by (0.1%) HgCl2 treatment for six minutes and sterile water wash, recorded 33% survival of cultures. The best explant for culture initiation was nodal segments. The best season for culture establishment was summer months (April-May) compared to rainy season (June- July). Loss of cultures was due to fungal contamination and necrosis of tissues. The media combination SH + Thidiazuron (TDZ) 0.03mg l-1 + Activated Charcoal (A.C.) 0.5% recorded bud expansion in 50% of the cultures within a period of nine days. Nodal segments are superior to shoot tips in culture establishment. Culture condition for culture establishment was 26 ± 20C at a light intensity of 1000 lux. The carbon source; 2% Sucrose + 1% glucose or 5% sucrose supported bud elongation and leaf expansion Refinement of culture establishment media was attempted with organic supplements; Coconut water (5, 10, 15 and 20% v/v), Casein hydrolysate (10, 25 and 50mg l-1) and Brassinolide (0.05, 0.1 and 0.2mg l-1) with nodal segments from juvenile seedlings, regenerants from coppiced trees and mature trees. In explants from juvenile seedlings and mature trees, 5 to 15%coconut water supported culture establishment. In juvenile explants, shoot elongation was also observed at 10% coconut water. Casein hydrolysate supported bud expansion in juvenile and mature tree explants at 10 to 50 mg l-1. Bud elongation and leaf expansion was observed at 25 mg l-1 concentration. Brassinolide (0.2 mg l-1) supported bud expansion in juvenile explants. The suggested media for explants from juvenile as well as coppiced trees for culture establishment of nodal segments of nutmeg is SH + 0.03 mg l-1 TDZ + 25 mg l-1 Casein hydrolysate + 2% sucrose + 1% glucose + 0.5% A.C. Casein hydrolysate concentration for explants from mature trees could be 50 mg l-1. In vitro seed germination was observed in mature seeds in presterilized bottles with water soaked cotton/ little water. Somatic embryos were formed at the cut portion of six month old seeds in the medium of ½ MS + 2% Sucrose + A.C. 0.5%. Proliferation of callus and somatic embryos was observed with the medium B5 + 0.1 mg l-1 Kin + 0.01 mg l-1 NAA + 0.01 mg l-1 GA3 + 10.0 mg l-1 Casein hydrolysate + . 0.5% A.C. The response was obtained two and a half months after inoculation. Three days old in vivo germinated seedlings did not established under in vitro condition even though surface sterilization treatment with 0.1% Emissan for thirty minutes followed by 0.1% HgCl2 for six minutes was given. Feasibility of grafting in juvenile plants was studied with epicotyl grafting and got 80% success. Grafting was done on twenty days old seedling with scion material from different seedling. In vitro epicotyl micrografting was done with in vitro raised scion and root stocks. It was also done on in vivo germinated seedlings after surface sterilization. Scion shoot of 2.5 cm length was grafted on twenty days old root stock. Graft was cultured in liquid nutrient medium and survived for two weeks. Later fungal contamination destroyed the cultures. Grafting with in vitro shoots on in vivo raised root stocks did not succeed
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    ThesisItemOpen Access
    Exploitation of invitro cultures of Indian Madder(Rubia cordifolia.Linn) for anticancerous compounds
    (Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2009) Labade, Dinesh Sitaram; KAU; Asha Sankar, M
    The present investigation on “Exploitation of in vitro cultures of Indian Madder (Rubia cordifolia L.) for anticancerous compounds” was carried out at the Plant Tissue Culture Laboratory of the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara and Amala Cancer Research Centre, Thrissur during the period 2006-2008. The study was undertaken with the objective to standardize the in vitro techniques for initiation and proliferation of static and suspension cultures of Rubia cordifolia and to screen the in vitro cultures for synthesis of naphthoquinone and quantify it. It was also envisaged to enhance the level of product synthesis in in vitro cultures and to assess the anticancerous activity of in vitro and in vivo extracts in terms of cytotoxicity, antioxidant and prooxidant activities in vitro. Leaf, nodal and root derived callus cultures of Rubia cordifolia were established in vitro. Explants were pre treated with the fungicide, Bavistin 2.5 per cent for 15 minutes. Surface sterilization with mercuric chloride (HgCl2) at 0.1 per cent for 1 min and 30 sec was effective for yielding healthy, contamination free cultures from nodal segments and leaves, respectively. MS medium at full strength, supplemented with NAA at 2 mg l-1 along with BA at 0.5 mg l-1 was observed ideal for initiation and proliferation of calli. The auxin synergist phloroglucinol, when supplemented to the medium, did not not yield encouraging results, with respect to callusing in the experimental species. Root derived cultures were inferior with respect to callus initiation and proliferation, registering low values for all the parameters studied. Incubating in vitro cultures under illuminated condition at 26 ± 2 C was superior to dark incubation, with respect to callus initiation and proliferation. Chloroform – methanol at 8.5 :1.5 ratio was indentified as the appropriate solvent system for detection of naphthoquinone on thin layer chromatograms in the test extracts of the experimental species, with alcoholic KOH (10 per cent) as the spray reagent. Ms medium at full strength, fortified with NAA and BA at 2.0 mg l-1 and 0.5 mg l-1 respectively, which recorded maximum naphthoquinone synthesis, was standardized as the production medium. Enhancing concentration of sucrose to 5 per cent in the production medium, did not elicit a positive response on naphthoquinone production in vitro. Reducing nitrate concentration of the production medium, to half and one fourth the original concentration, resulted in enhanced in vitro synthesis of the target compound. Supplementing the production medium with yeast extract (1 per cent and 2 per cent) as well as precursor feeding with phenyl alanine and tyrosine each at levels of 50 mg l-1, 100 mg l-1 and 150 mg l-1 exerted a favourable influence on synthesis of naphthoquonines, in vitro. Incubation in dark resulted in marginal increase in in vitro production of naphthoquinones. Incorporation of autoclaved mycelia of Pythium aphanidermeatum at levels of 2.0 per cent and 5.0 per cent resulted in enhanced in vitro production of naphthoquinone. The abiotic elicitor, salicylic acid at concentration of 10 μM and 100 μM resulted in maximum synthesis of naphthoquinones in in vitro root cultures (8.76 units g -1 calli) of Rubia cordifolia. Immobilization of test calli with sodium alginate – calcium chloride complex as well as subjecting the in vitro cultures to stress conditions, as imposed by sorbitol failed to bring about an enhancement in the in vitro production of naphthoquinones. None of the explants employed in the study induced hairy roots, when co- cultured with the Agrobacterium rhizogenes strains, MTCC 2364 and MTCC 532. Based on cell count, subculturing intervals of leafs, nodal and root derived suspension were fixed as 24, 27 and 27 days respectively with the respective packed cell volume as 0.93 per cent, 0.83 per cent and 0.80 per cent. Naphthoquinone was detected, in ex vitro and in vitro test extracts at all levels of maturity tested. Both ex vitro and in vitro root extracts exihibited maximum cytotoxicity, as revealed by the percentage of cell death on DLA and EAC cell lines as well as their IC50 values. As compared to whole plant extract, in vitro systems of the experimental species exhibited least antioxidant action. Extent of pro-oxidant activity was higher in in vitro root extract of the experimental species.
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    ThesisItemOpen Access
    Molecular characterization of katte mosaic virus of cardamom ( Elettaria cardmomum Manton )
    (Centre for Plant Biotechnology and Molecular Biology, College of Horticulture,Vellanikkara, 2016) Abida, P S; KAU; Manglam Arya
    Cardamom ( Elettaria cardamomum Maton) considered as “Queen of Spices” belongs to the family Zingiberaceae. Due to its aroma, cardamom is one among the most expensive spices in the world. Cardamom is infected by several fungal, bacterial and viral diseases. Katte or Cardamom mosaic disease is the most destructive viral disease affecting cardamom plantations worldwide. The disease is caused by Katte mosaic virus or Cardamom mosaic virus (CdMV) and spreads through infected suckers or through aphid Pentalonia nigronervosa Coq. The loss in yield due to katte disease ranges from 38 to 69 per cent and when infection occurs at early stage, the loss is complete. Management of katte disease totally depends on the use of disease free planting materials. As the infected plant often remains symptomless, identification and diagnosis of the virus becomes difficult at early stages. The present study was undertaken to develop serological and PCR based methods for identification and characterization of CdMV of cardamom. The infected and healthy samples were collected from four locations of Idukki and two locations of Wayanad districts of Kerala. The viral protein was isolated and purified from infected samples and SDS-PAGE analysis of purified protein from infected plants revealed the presence of 37 kDa band of viral coat protein. The purified protein from the infected plants was further used as antigen for the production of polyclonal antibody against CdMV in 6-9 month old rabbit. The rabbit was immunized with 5 mg of purified protein. The blood sample from the immunized rabbit was collected and the antibody was purified. The ODD (Ouchterlony Double Diffusion) assay was performed to standardize the titre of the antibody and results had shown that antigen-antibody complex was formed in 1:10, 1:100 and 1:150 dilutions of primary antibody. The indirect ELISA was carried out with 1:10, 1:100 and 1:150 dilutions of primary antibody and 1:200 dilution of secondary antibody. It was found that virus was easily detected in the crude extract of infected leaves with 1:100 dilution of primary antibody. Indirect ELISA was also performed for investigating the serological relationship of Katte mosaic virus with other viruses. The result of indirect ELISA revealed that the crude sap of infected cardamom leaves cross reacted with the antibody specific for Banana Bract Mosaic Virus (BBrMV) which also belongs to same potyvirus group. For the detection of CdMV through RT-PCR, the total RNA was isolated from the infected and healthy plants using Trizol and converted to cDNA. A total of 11 primers were designed for the amplification of coat protein gene of the virus using Primer 3 software. The primers designed were used for synthesis of second strand of cDNA and presence of virus was detected. Out of 11 primers, 8 primers were able to amplify the coat protein gene of the virus in infected plants. The band size of 250, 650, 750 and 950 base pairs were observed in infected plant but not in healthy plant samples. The viral amplicons of 250, 950, 750, 650 base pairs were generated with primers CP-2, CP-9, CP-10 and CP-11 respectively. These amplicons were further eluted, reamplified and sequenced. The nucleotide sequence annotated using bioinformatics tools BLASTn and BLASTx. The result of BLASTn showed 90 to 100 per cent nucleotide sequence similarity with CdMV whereas; the result of BLASTx revealed that the sequence had 55-100 per cent similarity with the coat protein of CdMV. The phylogenetic analysis was performed using 950 bp products generated with CP-9 primer. The phylogenetic tree was developed using MEGA.7 software by utilizing neibhourhood joining method. The result of phylogenetic analysis revealed that the isolates of Ambalavayal, Meppadi and Myladumpara are closely related and also related to isolates of Irettyar and Paravalam whereas, Pampadumpara isolate showed more variation to the above isolates. The methods developed in the present study are useful in detecting the Katte mosaic virus or Cardamom mosaic virus in the infected leaf samples of cardamom. The method will be useful in virus indexing, quarantine management in germplasm exchange, germplasm management, supply of disease free planting materials to the farmers and also for selecting the resistant line or cultivar for large scale production or incorporation in breeding programme for crop improvement.