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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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ThesisItem Open 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; KAUInvestigations 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.ThesisItem Open 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 MathewCowpea (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 directionThesisItem Open Access Genetic transformation of Amorphophallus paeoniifolius (Dennst) Nicolson(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2014) Leen Abraham, N; KAU; Makeshkumar, TA study on Agrobacterium-mediated genetic transformation of Amorphophallus paeoniifolius (Dennst.) Nicolson was conducted at the Central Tuber Crop Research Institute, Sreekariyam, Thiruvananthapuram during 2013- 2014. Calli were initiated using petiole and leaves of in vitro plantlets of elephant foot yam cv. Gajendra in callus induction media. After four weeks of incubation, actively dividing globular, hard and creamy white calli were developed. Subculture of developed calli was repeated periodically (20 days) in CIM with an approximate size of one cm2. 15 days old calli was found to be suitable for transformation study. Calli sufficient for the transformation study was obtained after 3 months of subculture. Experiments were conducted to evaluate the sensitivity of elephant foot yam calli to different doses of antibiotics viz. geneticin, hygromycin, ticarcillin. It was observed that complete death and discoloration of the calli obtained with 20 mgl-1 geneticin and 10 mgl-1 hygromycin from sixth week treatment. Statistical analysis of sensitivity response of calli indicated that LD100 was 20 mgl-1 and 5 mgl-1 with geneticin and hygromycin respectively. Sensitivity of the calli to ticarcillin was studied and the responses are analysed with ANOVA. The lowest lethal concentration of ticarcillin was found to be 650 mgl-1. So, concentration below 650 mgl-1 can be used for the successful elimination of Agrobacterium without affecting the regeneration potential of explant. 500 mgl-1 ticarcillin used in this study was observed sufficient for the successful elimination of Agrobacterium without affecting the regeneration potential of calli. For the optimization of parameters affecting transformation, experiments were conducted for the standardisation of optimum concentration of acetosyringone, time of co cultivation, temperature of co cultivation, and suitable Agrobacterium strain. In a study conducted for standardisation of optimum concentration of acetosyringone, increasing number of transformants was obtained with increase in acetosyringone. Significantly higher GUS staining of calli (21.5896) was achieved with the addition of 400μM acetosyringone in the co cultivation media. The effect of number of days of co cultivation on transformation was compared on the GUS expression of 14-day old selected calli. Two-three days of co-cultivation was determined to be the suitable for elephant foot yam because prolonged co-cultivation period (more than three days) was found to promote overgrowth of bacteria and subsequent death of the calli. Correspondingly the transformation percentage was found to decrease with the decrease (less than two days) of co-cultivation period. Investigation of the effect of temperature during co cultivation in elephant foot yam calli revealed that temperature plays an important role in transformation efficiency. Higher temperature, 28°C was found to be optimal to support the highest transient transformation frequency in elephant foot yam and dramatic transient expression reduction occurred when temperature decreased from 22 °C to 20°C. Transformation efficiency with respect to the different strain of Agrobacterium was investigated and the results showed that maximum percent of GUS stained tissue (24.5 percent) of transformants was obtained with the strain LBA4404 with pOYE153 vector followed by AGL0/pOYE153 (14 percent) and GV3103/pCAMBIA 1305.2 (6 percent). GUS assay of transformed callus showed blue colour and confirmation was done by PCR analysis with specific primers and southern blotting. PCR amplification of the DNA of the calli survived in selection medium yielded an expected band size of 280 bp for nptII primer, two bands of size 880bp and 700bp for GUS primer, 300 bp single band for hpt primer and GUSPlus primer. No amplification was obtained for untransformed calli DNA. Nucleic acid spot hybridisation of putative transformants of elephant foot yam further confirmation of the presence of transgene in the DNA. Hybridisation with nptII probe yield spots of varying intensity for all the transformants of AGL0/pOYE153 and LBA4404/pOYE153. Whereas only 5 out of the 8 transformants of GV3103/pCAMBIA1305.2 gave positive for hpt probe and the intensity of spot was low when compared to the spots obtained with nptII probe. Southern hybridisation with DIG labelled nptII probe yield a band for positive control (pOYE153 plasmid) whereas the bands in sample lane was not observed. It is possible that the concentration of DNA (10μl) used in the blot was too low for detection of T-DNA inserts. Hybridisation with hpt probe gave a single band corresponding to the putative transformants lane, which are visible after 30 min exposure indicated that successful hybridisation of the DIG-labelled hpt probe. But the absence of band for positive control was not expected.ThesisItem Open Access Genetic diversity analysis in taro (Colocasia esculenta (L.) Schott) of north east India(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2014) Vinutha, K B; KAU; Asha, Devi AThe study entitled “Genetic diversity analysis in taro [Colocasia esculenta (L.) Schott] of North East India” was carried out at the Division of Crop Improvement, Central Tuber Crops Research Institute, Sreekariyam, Thiruvananthapuram during 2013-2014. The objective of the study was to assess the genetic diversity among taro accessions from North East India, which is considered to be one of the centers of origin of taro, using morphological descriptors and SSR markers. The knowledge can be exploited in the heterotic breeding of taro to develop improved varieties suiting various needs. National and international germplasm repositories conserving root and tuber crops can also use the data to maintain taro germplasm efficiently. Twenty five accessions of taro collected from the various North Eastern states of India and maintained at CTCRI were selected for the study. Morphological characterization was performed at the maximum vegetative stage and tuber characters at harvest using ten quantitative and 28 qualitative traits. A combination of NBPGR and IPGRI descriptors were used to explain the wide range of morphological variability. The data was statistically interpreted in terms of diversity indices, PCA, ANOVA and cluster analysis using R statistical package and SAS program. The diversity indices (H’=0.87; D=1.00) revealed a high level of morphological diversity among the taro accessions. The first four components explained 76.59 per cent of the total variation with leaf margin colour, petiole colour (top 1/3rd, middle and base), leaf colour lower, sheath colour and sinus colour contributing maximum to the variability. ANOVA showed significant (P<0.01) variation for 7 out of the 10 quantitative traits studied. Duncan’s multiple range test gave a grouping based on the mean values of quantitative traits. Five major groups were revealed after hierarchical cluster analysis based on Euclidean distance, which did not bear any relation to the geographical origins of the accessions. A protocol was developed for the isolation of good quality DNA overcoming the high levels of secondary metabolites in taro. PCR conditions for SSR detection in taro were also optimized successfully. Ten out of 18 SSR primers were selected for the study after screening. Denaturing PAGE followed by silver staining was performed to analyze the variability among accessions at the molecular level. The average number of alleles and Shannon’s diversity index ranged from 6.0-12.57 and 1.59-2.37, respectively. The polymorphic marker ratio was found to be high for all the primers (0.76-1.0); however, Ce1 A06, Ce1 B03, Ce1 C06, Ce1 F04 and uq201-302 gave the maximum ratio of one. Cluster analysis based on Jaccard’s distance revealed five broad clusters which could not be correlated to the geographical similarities among the accessions. The parameters estimated from molecular and morphological characterization data established a high level of genetic diversity prevalent in the center of origin. The study revealed the absence of congruence between the clustering pattern and geographical origin suggesting that geographically diverse regions share ecologically similar characteristics and vice versa. Differences in morphological and molecular clustering patterns indicate the wide range of adaptations of the crop to the diverse environments inhabited. Though the Mantel’s test established no correlation (r = 0.1432; p = 0.0648) between the molecular and morphological distance measures, the study could identify two groups of accessions that clustered together in both the methods.ThesisItem Open Access Identification of AFLP marker linked with bacterial wilt resistance in chilli (Capsicum annum L)(Centre for plant biotechnology and molecular biology, College of horticulture, Vellanikkara, 2013) Thakur Pranita, Prabhakarrao; KAU; Deepu MathewChilli is one of the most important condiments in India and our country is the largest producer, contributing 25% of the world production. During 2010-11, India produced 0.8 mt of dry chilli from an area of 0.93 mha. Bacterial wilt (caused by Ralstonia solanacearum) is a major reason for the lower productivity of this crop, causing up to complete losses under severe infections. The study entitled “Identification of AFLP marker linked with bacterial wilt resistance in chilli (Capsicum annuum L.)” was carried out at the Centre for Plant Biotechnology and Molecular Biology (CPBMB), College of Horticulture during the period 2011-2013. The objective of the study was to identify AFLP marker linked with bacterial wilt resistance in chilli (Capsicum annuum L.). Three chilli genotypes Ujwala, Anugraha and Pusa Jwala and their progenies were used in the study. Anugraha is a chilli variety with resistance to bacterial wilt disease. Pusa Jwala is the near isogenic line (NIL) of Anugraha, both differing only in the gene for resistance to bacterial wilt. Ujwala was the donor parent for resistance while developing Anugraha from Pusa Jwala through back crossing programme. Pusa Jwala and Anugraha varieties were screened in open field with artificial inoculation to confirm the disease reaction. Highly resistant Anugraha plant was crossed with the pollen from most susceptible Pusa Jwala plant. The F1 seeds were harvested and this generation was field screened to observe the disease reaction. Previous report on bacterial wilt resistance points to monogenic homozygous recessive condition for resistance. Accordingly, all the F1 plants are supposed to be susceptible; but our screening had shown the resistance in F1 generation to be nearly 50 per cent. The reason for the deviation from the expected ratio is attributed to the selection of a heterozygous (Rr) plant as male parent. Further the F1 plants were raised in pots, selfed seeds were harvested and further the pots were infected with bacteria and F2 seeds harvested from susceptible F1s were used for raising the F2 population. F2 population was used as segregating population for generating the susceptible and resistant bulks in bulk segregant analysis (BSA) using AFLP method. Screening of 200 F2 plants was done along with parents Anugraha and Pusa Jwala using leaf cutting and pin pricking methods. F2 segregating plants have shown 69.5 per cent wilt incidence, pointing to ~3:1 ratio, confirming that the resistance is governed by homozygous recessive condition. DNA extraction was done from parents and all the 200 F2 plants and Ujwala by CTAB method (Rogers and Bendich, 1994). Good quality of DNA with UV absorbance ratio (A260/280) ranged 1.8- 2.0 were used for further AFLP analysis. DNA from 9 highly susceptible and 9 highly resistant F2 plants was separately pooled for developing bulk. BSA was carried out with AFLP analysis the DNA using resistant parent (Anugraha), susceptible parent (Pusa Jwala), resistance donor (Ujwala), F2 resistant bulk and F2 susceptible bulk. The AFLP was performed using the standard kit provided by Chromus Biotech, Bangalore, following the restriction digestions using frequent and rare cutters, adapter ligation, pre-amplification and selective amplification using the primer combination EcoACT+ MseCAC. The amplified PCR products were separated by capillary electrophoresis on an ABI Prism 310 Genetic Analyzer along with GeneScan™ 500 LIZ® size standard and data generated were collected by Data Collection 2.1 software. Totally, 124 bands ranging 50-500bp were amplified. Among them, three polymorphic bands with 103, 118 and 161 bp linked with the resistance allele and three polymorphic bands with 183, 296 and 319 bp linked with susceptible allele were identified. Due to the repeatable nature of AFLP, these 6 markers could be directly employed in MAS breeding programmes.ThesisItem Open Access Proteome analysis of induced systemic resistance medicated by plant growth promoting Rhizobacteria (PGPR) in rice for biotic stress(Centre for plant biotechnology and molecular biology, College of horticulture, Vellanikkara, 2013) Shinde Subhashini, Ganesh; KAU; Abida, P SRice (Oryza sativa L.), is vulnerable to a number of pest and diseases. Among them sheath blight disease caused by Rhizoctonia solani, and insect pest, brown planthopper (BPH) (Nilapavata lugens) are the most devastating agents and major challenge to rice cultivation. Approximately 30 per cent yield loss has been reported due to above said disease and pest. In this context, the present study entitled „proteome analysis of induced systemic resistance mediated by plant growth promoting rhizobacteria (PGPR) in rice for biotic stress‟ was carry out at the Centre for Plant Biotechnology and Molecular Biology (CPBMB), College of Horticulture, Vellanikkara during the period 2011-2013, with the objective to identify and characterize the interacting proteome in inducing systemic resistance in rice mediated by PGPR during pathogen and insect attack. Plant growth-promoting rhizobacteria (PGPR) are associated with plant roots and augment plant productivity and immunity. However, recent work by several groups shows that PGPR elicit physical or chemical changes related to plant defense, a process referred as „induced systemic resistance‟ (ISR). ISR induced by PGPR has suppressed plant diseases caused by a range of pathogens in both greenhouse and field (Yang et al., 2009). Jyothi (PTB -39), a popular rice variety of Kerala and susceptible to blight and BPH was used as the experimental material. The pot culture experiment was carried out by direct sowing. The biometric observations were taken on control plants and the plants treated with Pseudomonas fluorescens Pf1 (KAU culture) 30 days after sowing. Significant increase was observed in shoot length, root length, number of tillers, fresh weight and dry weight in Pf primed rice plants. Both R. solani and BPH were inoculated forty five days after sowing, to check the efficacy of Pf strain against the pathogen and insect. Protein analysis was carried out to study the molecular mechanisms operating behind the PGPR mediated pest and disease resistance and growth promotion. Total Proteins were isolated from rice plants treated with and without Pf and challenged with pest and pathogen at 0, 6, 24, 48, 72, and 96 hours after inoculation (HAI). The protein samples were analyzed by SDS-PAGE gel system. The comparison analysis of relative abundances of protein bands between inoculated and non inoculated samples were carried out. At 24, 48, and 72h, 16 proteins were expressed in Pf treated and control plants challenged with R. solani. A twenty nine kDa protein was up regulated in all the Pf treated plants challenged with R. solani and this was selected for sequencing. In all the Pf treated and BPH challenged plants 3 different proteins of 19, 23 and 30 kDa were expressed. Two protein bands of 19 kDa from 48h and 30 kDa from 96h were selected for MALDI-TOF sequencing. These three proteins bands were sequenced by MS MALDI-TOF in Sandor Proteomics, Hyderabad. Protein band 1 of 29 kDa, showed, 98 per cent homology to chloroplastic aldolase, fructose-bisphosphate aldolase and 81 per cent homology to peroxidase. The protein band 2 of 19 kDa, showed 84 and 86 per cent homology to 2- cys peroxiredoxin bas and 2-cys peroxiredoxin bas1, respectively. The Protein band 3 of 30 kDa, showed 99 per cent homology to small subunit of ribulose-1, 5-bisphosphate carboxylase and 100 per cent similarity to hypothetical proteins Os12g0291400 and OsI_38046. Western blot analysis was also carried out to further confirm the presence of PR protein chitinase. The analysis confirmed the presence of chitinase of molecular weight 17 kDa, 18 kDa, and 35 kDa in PGPR primed plant challenged with R.solani. The PO assay was also carried out to check the activity of peroxidase enzyme in both control and PGPR primed plants challenged with BPH and pathogen. PO activity was increased from 6 to 96 h after challenge inoculation in Pf primed plants. There was 43.62 and 21.9 per cent increase in PO activity over control plants in pathogen inoculated and BPH challenged Pf primed plants at 96hours. The result obtained gave information for further elucidation of candidate genes operating in signal transduction pathways mediated by PGPR during ISR to biotic stress.ThesisItem Open Access DNA fingerprinting of released varieties and selected superior somaclones of ginger (Zingiber officinale Rosc)(Centre for plant biotechnology and molecular biology, College of horticulture, Vellanikkara, 2013) Pujaita Ghosh; KAU; Shylaja, M RGinger (Zingiber officinale Rosc.), one of the widely cultivated and consumed spices worldwide, is well known for its medicinal properties also. As the natural variability stands limited in the crop, induction of variability through tissue culture techniques was attempted at College of Horticulture, Vellanikkara from 1996 onwards. After indepth investigations on the somaclones regenerated, two varieties viz., “Athira” and “Karthika” were released during 2010 and four clones viz. B3, 292R, 88R and 478R were selected as superior somaclones. For the newly released ginger varieties and selected superior somaclones in pipeline for release, no fingerprint data are available for genotype identification and protecting the plant varieties / clones. The investigations on “DNA fingerprinting of released varieties and selected superior somaclones of ginger (Zingiber officinale Rosc.)” were carried out at the Centre for Plant Biotechnology and Molecular Biology (CPBMB), College of Horticulture, Kerala Agricultural University, Thrissur during the period from January 2012 to March 2013. The objectives of the study were to characterize two released varieties and four selected superior somaclones using molecular markers and to develop a DNA fingerprint specific to each variety / somaclone. Morphological characters like growth habit and size and shape of the rhizomes were found to vary in the varieties / somaclones studied. The somaclone 292R could be distinguished based on its dwarf plant stature and dark green leaves. The variety Athira has bold and flat rhizomes while the variety Karthika has medium bold and round rhizomes. Quantitative clustering for vegetative and rhizome characters attempted as per Mahalanobis D2 analysis could group the varieties and somaclones into three separate clusters. Of the seven vegetative characters analysed, plant height and number of tillers showed more divergence. The number of fingers, girth of primary and secondary fingers, thickness of flesh and inner core were the characters which exhibited more divergence for the rhizome characters. For molecular characterization, good quality genomic DNA extracted from ginger varieties / somaclones using CTAB (Rogers and Bendich, 1994) method was used. Thirty five RAPD and thirty ISSR primers were screened for amplification of genomic DNA and ten RAPD and eleven ISSR primers were selected based on the amplification pattern. DNA fingerprints of the varieties / somaclones were developed utilizing the clear, distinct bands generated in RAPD and ISSR profiles and size of the bands. Different colour codes were assigned for sharing of bands between varieties / clones to generate specific fingerprints. The RAPD marker system could bring out unique bands in the variety Karthika and somaclones B3, 292R and 478R. The RAPD primer, OPA 12 produced unique band in Karthika and B3, the primer OPA 04 in 292R and the primer OPA 28 in 478R. ISSR marker system could also bring out unique band in the variety Athira with primer ISSR 06. The RAPD, ISSR and combined fingerprints developed for each variety / somaclone were unique. Variability in the somaclones and the extent of variability from source parent cultivars were analysed using cluster analysis. The dendrogram seperated Maran and Rio-de-Janeiro somaclones in two separate clusters. Somaclones derived from cultivar Maran exhibited more variability than somaclones from Rio-de- Janeiro. The variety Athira was more diverse from the source parent cultivar Maran. Similarly, the somaclone 292R was more diverse from the source parent cultivar Rio-de-Janeiro. The Resolving Power (Rp) of RAPD and ISSR primers ranged from 6.00 to 16.25, indicating the ability of the selected primers to distinguish the varieties / clones most efficiently. The Polymorphic Information Content (PIC) ranged from 0.67 to 0.88, indicating the suitability of the selected primers for DNA fingerprinting. RAPD, ISSR and combined fingerprints developed specific for the ginger varieties / somaclones could be utilized for registration, documentation of varieties and for settling IPR issues.ThesisItem Open Access Variability analysis in ginger (Zingiber Officinale Rosc) somaclones using molecular markers(Centre for plant biotechnology and molecular biology, College of horticulture, Vellanikkara, 2013) Gavande Sharda, Shivaji; KAU; Shylaja, M RGinger (Zingiber officinale Rosc.), an important spice crop grown in India, is much valued for its flavour and medicinal properties. As natural variability available in the crop is limited, somaclonal variation is being utilized for crop improvement programmes. Currently, molecular marker techniques are widely employed to detect and assess somaclonal variation in several crop species as they are stable, detectable in all tissues and are not confounded by environment, pleiotropic and epistatic effects. The present investigations on “variability analysis in ginger (Zingiber officinale Rosc.) somaclones using molecular markers” were carried out at the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Kerala Agricultural University during February 2012 to May 2013. The objectives of the investigations were to assess somaclonal variation in ginger at molecular level, to study the influence of genotype and mode of regeneration on somaclonal variation, to assess the extent of variability in somaclones from the original source parent cultivars and to select the variants. Two molecular marker systems viz. Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeats (ISSR) were utilized for the analyses. Ginger somaclones (180 Nos.) regenerated through various modes of regeneration viz. bud culture, indirect organogenesis / embryogenesis and in vitro mutagenesis, along with two source parent cultivars (Maran and Rio-de-Janeiro) were used for the present study. The genomic DNA was extracted from somaclones using CTAB method (Rogers and Bendich, 1994) and Sigma’s GenEluteTM Plant Genomic DNA Miniprep kit. The somaclones were grouped as per genotype and mode of regeneration. DNA extracted from individual somaclones was bulked as per the procedure reported by Dulson (1998). Bulked DNA samples of the thirteen groups of somaclones along with two source parent cultivars were subjected to RAPD and ISSR analyses with selected primers. Of the 35 RAPD primers screened, twelve gave good amplification. RAPD analysis using selected primers produced 129 amplicons, 44 were polymorphic with an average of 3.66 polymorphic bands / primer and a polymorphism percentage of 34.10. In ISSR assay, twelve selected primers produced 122 amplicons, 32 were polymorphic with an average of 2.66 polymorphic bands / primer and a polymorphism percentage of 26.23. The study could identify certain specific RAPD and ISSR primers for identification of Maran and Rio-de-Janeiro cultivars and irradiated mutants from non- irradiated somaclones. The dendrograms generated based on RAPD and ISSR profiles grouped the somaclones into two separate clusters, with somaclones of Maran in first subcluster of cluster I and somaclones of Rio-de-Janeiro in second subcluster of cluster I. The regenerants from Rio-de-Janeiro calli irradiated with 20 Gy and somatic embryogenic calli irradiated with 10 Gy formed the second cluster. RAPD and ISSR marker systems showed that somaclones derived from cultivar Maran exhibited more variability than Rio-de-Janeiro. RAPD marker system was more effective for bringing out variability. The variability observed in RAPD assay was 28 per cent while in ISSR assay it was 21 per cent and in the combined it was 25 per cent. In groupwise variability analysis using bulked DNA, the groups RC20 Gy and RSe10 Gy recorded higher variability from source parent cultivar. The variability exhibited in plantwise analysis using three selected primers (OPA 28, S11 and ISSR 05) was found very high (39%) as compared to groupwise analysis (25%). The somaclone RC2Kr1031 of the callus regenerants and RSe1Kr1052 of the somatic embryo regenerants showed more variability exhibiting 59 and 53 per cent variability respectively from the source parent cultivar Rio-de-Janeiro. The extent of variability in ginger somaclones could be assessed using molecular markers and in vitro mutagenesis could be employed to widen the genetic base in ginger.ThesisItem Open Access Molecular characterization of male sterility in ridge gourd (Luffa acutangula (L.) Roxb.)(Centre for plant biotechnology and molecular biology, College of horticulture, Vellanikkara, 2014) Sonwane Shital, Marotirao; KAU; Deepu, MathewThe development of hybrids with desirable heterosis is a major goal in plant breeding. In traditional hybridization, following the emasculation which is a labour intensive process, pollen is manually transferred to the female organs. The higher cost of the hybrid seeds, considerably owing to the labour costs for emasculation, is the major constraint in achieving more rapid adoption of vegetable hybrid technology. Use of male sterile parent is understood to reduce the cost of hybrid seed production by avoiding the manual emasculation. In this context, the recently reported male sterile line of ridge gourd from Kerala Agricultural University, is commercially very important. A system to confirm the male sterility at an early phase of parent itself, is very important since any kind of fertility restoration at a later phase will fail the hybridization programme. A molecular marker associated with the male sterility will be enormously useful at this phase, to confirm the male sterility of the female parent used in the hybridization programme. The study entitled “Molecular characterization of male sterility in ridge gourd Luffa acutangula (L.) Roxb.” was carried out at Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Kerala Agricultural University, with the objective to identify the molecular marker/s linked with the male sterility in the newly reported sterile line of ridge gourd from KAU, using SSR and ISSR marker systems. Plant materials used in this study were three male fertile lines, Haritham, Deepthi, and Arka Sumeet and the Haritham (male sterile line of KAU). In the male sterile line, unopened rudimentary male buds with poorly developed anthers containing shrunken, non viable pollen grains were observed. In male fertile plants, the anthers were well developed with viable pollen grains. Total genomic DNA was extracted from the young leaves, using CTAB method and SSR and ISSR marker systems were employed for characterizing the male sterility. SSR primers are highly genus specific and since genomic data on Luffa is lacking, suitability of SSR primers from the related genus such as Citrullus and Cucumis was attempted. Initial attempts on electrophoresis of SSR amplicons on 2 per cent agarose gel were successful for few primers only. Thus, to resolve the small fragments, 10 per cent native PAGE was subsequently used. In SSR assay 25 primers were screened, among which 14 were selected. Total genomic DNA of the male fertile and sterile lines were amplified with these SSR primers. The primers CI1-21, DE0144 and CsWCT25 have yielded distinctly polymorphic bands associated with the male sterility with 125, 50, 350 bp sizes, respectively. In ISSR assay, among the 49 primers screened initially, 16 were selected for the study. UBC841 has yielded a distinctly polymorphic band for male fertility at 1185bp. This marker was missing in the male sterile line. Dendrogram was generated based on the ISSR study in order to study genetic relationship among the ridge gourd lines. Haritham (fertile) and Haritham (male sterile) showed 82 per cent similarity. The universal mitochondrial primers were additionally screened in this study and among them; Rps14 gave polymorphic band linked with male sterility, at 80 bp size. Direct sequencing with NGS platform was done of the identified polymorphic bands, it was failed with CI1-21, Rps14, DE0144 and UBC841. Only with CsWCT25 sequence of 225 bp was obtained. CsWCT25 is already reported to be tightly linked with gynoecy in cucumber, SCAR primers are also designed using this sequence. These markers will have wide application in marker assisted selection and hybrid seed production.
