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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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201424
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Subject: Plant Biotechnology × Author: KAU × End date: 2014 × Start date: 2014 × Type: Thesis × Thesis Type: M.Sc ×
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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
    Genetic transformation of Amorphophallus paeoniifolius (Dennst) Nicolson
    (Department of Plant Biotechnology, College of Agriculture, Vellayani, 2014) Leen Abraham, N; KAU; Makeshkumar, T
    A 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.
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    ThesisItemOpen 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 A
    The 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.
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    ThesisItemOpen 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, Mathew
    The 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.
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    ThesisItemOpen Access
    Induction of variability in anthurium (Anthurium andreanum Lind.) through in vitro mutagenesis
    (Centre for plant biotechnology and molecular biology, College of horticulture, Vellanikkara, 2014) Yashawant Kumar, Srivastava; KAU; Nazeem, P A
    Anthuriums (Anthuriumandreanum Lind.) of family Araceae, is highly valued for their exotic flowers and foliage. The attractivecharacteristics like vibrant inflorescence with straight spathe, candle-like spadix, exotic foliage and particularly the long lasting ‘flower’ of anthurium have ensured its great commercial importance. Anthurium is conventionally propagated by traditional vegetative methods such as stem cuttings and suckers which are tedious and not practical when carried out on a large scale. Plants derived from seeds showmarked variation in colour, quality, yield and time of first flowering. Seedviability and germination percentage are also low. Seed are viable only for two to three days and germination is as low as 20 to 30 per cent. Hence, there is a needto standardize a quicker method of propagation which may be achieved throughin vitro techniques.Tissue culture greatly increases the normal multiplication rate of plants and can provide a source of clean planting material. Vegetative means of propagation and poor seed viability limits genetic variation in anthurium and this necessitate alternate means to wider the genetic base. Invitromutation breeding is therefore being proposed as a means to create additional variation. The present study entitled “Induction of variability in anthurium (Anthurium andreanum Lind.)throughin vitro mutagenesis” was conducted with the objective to induce variation in anthurium var. Tropical and to characterise the variability through morphological and molecular assay. In vitro mutagenesis in anthurium was carried out using invitro cultures of var. Tropical as source material for treatments. Cultures were treated with gamma radiation as physical mutagen at different doses such as 5, 10, 15 and 20 Gy and chemical mutagen Ethyl Methane Sulphonate (EMS) at different concentrations such as 0.1, 0.2 and 0.5 per cent for 30 minutes. The irradiation was carried out at Radio Tracer Laboratory, College of Horticultureat room temperature using a gamma chamber equipped with 60Co source. After irradiation the cultures were kept in dark room for two days and later it was transferred to fresh media and incubated in light. The cultures were observed periodically for their response. Cultures exposed to higher doses lost their green appearance while the culture at lower doses (5 Gy) were good in multiplication and number of shoots compared to control. The cultures were sub cultured at one month interval; rooted plantlets planted out and hardened. The cultures differed in their in vitro response with respect to plant height, number of leaves, leaf dimensionsand flowering habit. Plantlets derived from low doses (5 Gy) of irradiation performed better than control while higher doses (15 to 20 Gy) gave stunted plants with more number of leaves. The cultures treated with EMS failed to regenerate except those with 0.1% treatment. Mutated plants started flowering within seven months and lot of variations were observed with respect to flower size, colour, length of flower stalk, arrangement of spathe and spadix. The selected variants were analysed at molecular level using RAPD and ISSR assay and genetic variation was confirmed. The desirable mutants are to be further evaluated for their stability with respect to the altered trait.
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    ThesisItemOpen Access
    Identification of molecular marker linked to the resistance for vascular streak dieback disease in cocoa (Theobroma cacao L.)
    (Centre for plant biotechnology and molecular biology, College of horticulture, Vellanikkara, 2014) Ekatpure Sachin, Chandrakant.; KAU; Deepu Mathew
    Cocoa is the third important plantation crop next to coffee and tea and is the third highest traded commodity in the world after coffee and sugar. Current world production of cocoa is about 4 million tonnes but India contributes only 0.02 per cent. In Kerala, area under the cocoa is 12,480 ha. with the production of 6.14 metric tonnes and productivity 490 kg/ ha (Directorate of Cashewnut and Cocoa Development, 2013). Vascular streak dieback (VSD), caused by the fungus Oncobasidium theobromae and first reported in India in 1981, is a very serious disease of cocoa that has already spread to all the cocoa growing tracts of India. The characteristic symptoms include a green-spotted chlorosis and fall of leaves beginning on the second or third flush behind the stem apex, raised lenticels, and darkening of vascular traces at the leaf scars and infected xylem. Eventually complete defoliation occurs and, if the fungus spreads to the trunk, the tree will die. The confirmation of transfer of gene is at most important in crop breeding and availability of a tool to quickly detect the gene will reduce the breeding cycle length. Molecular markers are already proven good to detect the genes of interest. The present study aims at developing a reliable molecular marker linked with the gene offering resistance to VSD in cocoa, using SSR and ISSR marker systems. Extensive germplasm collection and evaluation have been done at Kerala Agricultural University, India and tolerant cocoa clones were identified. Using these clones, 45000 hybrids have been bred and were screened for resistance using natural inoculum under high humidity and 1177 field established resistant hybrids are being evaluated for the past 15 years. Already, KAU has released three hybrids and 7 selections of cocoa with field tolerance to VSD. In the present investigation, twelve genotypes of cocoa, having different responses to VSD namely, VSD I-4.6, VSD I-4.11, VSD I-5.8, VSD I-6.9 (resistant), G VI-50, G VI-52, G VI-82, G VI-100 (Susceptible), G VI-25, G VI-53, G VI-67 and G VI-144 (partially resistant) were employed. The genotypes were selected on the basis of field screening experiments started in 1998-1999 at Cocoa Research Station, Kerala Agricultural University. Genomic DNA isolated using modified CTAB method suggested by Doyle and Doyle (1987). ISSR assay on the total genomic DNA of twelve cocoa clones using 71 primers had shown that the primers UBC811, UBC815, UBC826, UBC857 and UBC866 are capable to yield the polymorphic bands, in relation to VSD resistance, whereas, SSR assay with 46 primer sets has failed to generate any marker for the same. The most distinct polymorphic marker generated in the resistant lines by the ISSR primer UBC857 was eluted. This DNA was further subjected to PCR analysis using the same primer, to verify its suitability for direct sequencing. The direct sequencing has yielded 246 nucleotides, which on BLASTn had shown 94 per cent identity with the Theobroma cacao microsatellite DNA clone of mTcCIR42 SSR (NCBI accession number AJ271944). Subsequently, the clones were subjected to SSR assay using the primer mTcCIR42. The assay has generated distinctly polymorphic banding pattern that differentiated the resistant lines from the susceptible and partially resistant ones. Since highly successful in differentiating the resistant, susceptible and partially resistant cocoa clones, these markers are recommended for use in marker assisted breeding for VSD resistance.
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    ThesisItemOpen Access
    Morphological and molecular characterization of byadagi chilli (Capsicum Annum L.)
    (Centre for plant biotechnology and molecular biology, College of horticulture, Vellanikkara, 2014) Renuka S, Karennavar.; KAU; Shylaja, M R
    Byadagi is a famous chilli type grown in Karnataka and is named after the town “Byadagi” which is located in Haveri district of Karnataka. This chilli is known for its deep red colour and negligible or zero pungency. The demand for Byadagi chilli has increased enormously as a source of natural red colour in food industry. Spices Board has taken steps to protect Byadagi chilli under GI registration. The investigations on “Morphological and molecular characterization of Byadagi chilli (Capsicum annuum L.)” was carried out at the Centre for Plant Biotechnology and Molecular Biology (CPBMB), College of Horticulture, Kerala Agricultural University, Thrissur during July 2013 - June 2014. The study was aimed to characterize Byadagi chilli using morphological and molecular markers with special emphasis on colour and pungency. Two Byadagi chilli cultivars viz. Byadagi Kaddi and Byadagi Dabbi and two KAU released varieties viz. Ujwala and Anugraha were characterized in the study. The variety Ujwala was selected for its high pungency and variety Anugraha for its less colour on drying. Morphological characterization was done as per NBPGR minimal descriptor for chilli. Molecular characterization was done using two different marker systems viz. RAPD and ISSR. Twenty one qualitative and sixteen quantitative characters were recorded as per minimal descriptor. Byadagi cultivars possessed dark green medium sized leaves, dark red ripened fruits with wrinkled surface, higher fruit weight (6.27 g), less number of fruits per plant (17.21), more number of seeds per fruit (97.49) and lesser fruit yield (100.12 g). KAU varieties viz. Ujwala and Anugraha recorded more number of fruits per plant (88.08) with less fruit weight (1.79 g) and higher fruit yield (152.4 g) with red coloured ripened fruits with smooth surface. The dried fruits were analysed for colour and pungency at Spices Board quality evaluation laboratory, Kochi. Byadagi cultivars recorded higher colour value (108.92 ASTA) and lower pungency (0.045 per cent capsaicin) while KAU varieties viz. Ujwala and Anugraha possessed lower colour value (59.1 ASTA) and higher pungency (0.32 per cent capsaicin). The RAPD marker system could generate a total of 155 amplicons with fourteen primers exhibiting an average polymorphism of 65.23 per cent and 7.14 polymorphic amplicons/ primer. The ISSR marker system could generate a total of 103 amplicons with eleven primers exhibiting an average polymorphism of 61.35 per cent with six polymorphic bands per primer. The dendrogram generated with RAPD, ISSR and combined profiles grouped Byadagi cultivars and KAU varieties in separate clusters. The varieties Ujwala and Anugraha were found closer with 67 per cent similarity and Byadagi Kaddi and Byadagi Dabbi with 61 per cent similarity. Eight amplicons resolved with five primers (S12, S13, OPAH 06, OPA10 and OPE18) in RAPD marker system and twelve amplicons resolved with seven primers (2UBCS2, ISSR05, ISSR08, ISSR15, UBC823, UBC840 and 17899A) in ISSR marker system were found specific to Byadagi Kaddi and Byadagi Dabbi. The morphological and molecular characterization attempted in the present study could distinguish the Byadagi cultivars from the KAU varieties , Ujwala and Anugraha with respect to colour and pungency. The specific traits of Byadagi cultivars like high colour and low pungency are expressed in a similar fashion under Kerala conditions also. In depth investigations on the unique amplicons identified specific to Byadagi cultivars will give further insight into the genes involved in colour and pungency of chilli, which could be utilized further in marker assisted selection, metabolic pathway engineering and secondary metabolite production.
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    ThesisItemOpen Access
    Micropropagation of gerbera (Gerbera jamesonii Bolus) and assessment of genetic stability of plantlets using ISSR assay
    (Centre for plant biotechnology and molecular biology, College of horticulture, Vellanikkara, 2014) Awchar Datta, Manikrao; KAU; Shylaja, M R
    Gerbera jamesonii Bolus commonly known as African daisy is an important cut flower ranking fifth in the global cut flower trade. Gerbera is also used as potted plant due to its attractive flowers of varying colours. It is generally propagated by division of suckers or clumps, but the multiplication rate is found very slow. New varieties are introduced every year in large numbers for commercial cultivation through high-tech system. Development of an efficient micropropagation protocol is of great significance for meeting the large scale demand of quality planting material and to popularise the new varieties. As there is high demand for new variants in gerbera, tissue culture induced variability also could be exploited in breeding programmes. The investigations on micropropagation of gerbera (Gerbera jamesonii Bolus) were hence taken up at Centre for Plant Biotechnology and Molecular Biology, College of Horticulture from 2012 to 2014. The present study aims to micropropagate gerbera using flower buds and leaf explants and to test the genetic stability of the micropropagated plants using ISSR assay. The IIHR variety Arka Krishika and three other varieties procured from AVT, Kochi viz., Dubai, Shania and Hotpsring were utilised for the study. Micropropagation protocols were attempted in different gerbera varieties using two different pathways viz., direct and indirect organogenesis with explants like flower bud and leaves. The micropropagation protocol developed at CPBMB by Shylaja et al. (2014) was used to regenerate plants from flower bud explants. Regeneration from flower bud explants was achieved through direct organogenesis in three gerbera varieties viz., Arka Krishika, Dubai and Shania. An efficient protocol was standardised from in vitro leaf explants through indirect organogenesis and from initial cultures to establishment of micropropagated plants it took only six months. High genotypic difference was observed in propagule multiplication in different gerbera varieties studied. The white flowered variety Dubai showed high rate of multiplication in both the routes of micropropagation viz., indirect organogenesis using leaf explants and direct organogenesis using flower bud explants. The potting media for hardening micropropagated plants were standardised and plantlets produced were successfully acclimatized. Genetic stability studies using ISSR assay were carried out in three groups of plants viz., mother plants, flower bud regenerants and leaf calli regenerants. Of the seven gerbera specific ISSR primers tested, five primers showed monomorphic banding pattern. In variety Dubai, primers ISSR 18 and ISSR 21 exhibited polymorphism to the extent of 25 to 33.33 per cent. In variety Shania, the primers ISSR 15 and ISSR 25 exhibited polymorphisms to the extent of 25 to 33.33 per cent. Genetic stability studies showed that mother plant and regenerants derived from flower buds were uniform to the extent of 80 per cent. Variation at DNA level observed was more (40%) for plantlets regenerated through indirect pathway. The established micropropagated plants (200 Nos.) and plants showing variation at DNA level are to be evaluated further for flower production and floral characters before recommending the protocol for commercial micropropagtion. Genotype specific optimisation should be done in micropropagation protocol for effective large scale multiplication of different varieties.
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    ThesisItemOpen Access
    Molecular characterization and DNA fingerprinting of selected cashew (Anacardium occidentale L.) varieties of KAU
    (Centre for plant biotechnology and molecular biology, College of horticulture, Vellanikkara, 2014) Purushottam Meena; KAU; Abida, P S
    Cashew (Anacardium occidentale L., Anacardiaceae), a foreign exchange earning horticultural plantation crop, native of north east Brazil and was introduced into India by Portuguese travellers during 16th century. Since its introduction, cashew has very well adapted to the Indian climatic conditions and is grown both in the east coast and west coast regions. India was the first country to exploit the international trade of cashew kernels in the early part of 20th Century. Now India is the largest producer, processor, consumer and exporter of cashew in the world. It is famous for its delightful nutritious kernels and apple, and it by-product like cashew nut shell liquid (CNSL), is commercially used in polymer and paint industries. More than 46 high yielding varieties of cashew released in India, Kerala Agricultural University holds the credit for 16 varieties. The varieties selected for this study include three hybrids (Poornima, Priyanka and Dhana) and two selections (Sulabha and Madakkathara-1) based on their unique characteristics such as Poornima with medium nut size, best shelling percentage (31%), Priyanka large sized nut and yellowish red apple, Dhana large sized nut, better shelling percentage, and wide acceptability, Sulabha large sized nut and kernel, light orange colour apple and late flowering, Madakkathara-1 for small sized nut and early flowering. The present study entitled ―Molecular characterization and DNA fingerprinting of selected cashew (Anacardium occidentale L.) varieties of KAU‖ was carried out at the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara during the period 2012-2014. The objective of the study was to characterize the five selected cashew varieties of KAU using ISSR and SSR molecular markers, and to develop a DNA fingerprint specific to each variety. Morphological characters were recorded from three mature plants per variety of almost same age, at Cashew Research Station, Madakkathara as per the IBPGR minimal descriptor for cashew. Grafts of selected cashew varieties were obtained from Cashew Research Station, Madakkathara and maintained at CPBMB, College of Horticulture. Genomic DNA of good quality was extracted from cashew genotypes using CTAB method (Rogers and Bendich, 1994) with slight modifications. PCR components and thermal profiles were optimized for ISSR and SSR assay. Thirty nine ISSR primers and twenty five SSR primer pairs were screened for amplification of genomic DNA out of them, eleven each of ISSR and SSR primers were selected for further analysis, based on the reliable distinct amplification pattern. In ISSR and SSR analyses, selected primers produced total of 149 and 19 amplicons, respectively. Out of which 69 and 12 were polymorphic with an average of 6.27 and 1.09 polymorphic bands per primer and polymorphic percentage were 46 and 63, respectively. The resolving power (Rp) worked out for the different primers ranged from 1.4 to 7.0 in ISSR analysis, indicating the capacity of the primers selected to distinguish the varieties. The polymorphic information content (PIC) ranged from 0.256 to 0.42 for ISSR analyses indicating the variability among the genotypes. While, the PIC value for SSR primers ranged from 0 to 0.50. The ISSR and SSR banding pattern was scored and analysed for their similarity, using the software NTSys pc (version 2.02i). The dendrogram generated with ISSR, SSR and combined profiles grouped the selected cashew varieties in separate clusters. The varieties Priyanka and Dhana were found more closer with 98 per cent similarity but a field grown graft of Poornima was the most distinct one from other cashew genotypes. Distinct amplicons developed through ISSR and SSR analyses were used to develop the DNA fingerprint of cashew genotypes. Sharing of amplicons for each primer with other genotypes was also analyzed and demarcated with different colour codes in the fingerprint developed. In ISSR assay, each primer gave a unique amplification pattern for each variety. Separate fingerprints were developed for all the five varieties through ISSR and SSR analysis. The DNA fingerprints developed could be utilized for the variety identification and to solve IPR issues.