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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 Immunological and molecular detection of banana viruses and production of disease free planting materials(Department of Plant Pathology, College of Agriculture, Vellayani, 2014) Aliya, Ferzana; KAU; Umamahesaran, KThe study entitled "Immunological and molecular detection of banana viruses and production of disease free planting materials" was conducted in College of . Agriculture, Vellayani, and Thiruvananthapuram during !he period of2011-2014. Symptomatological studies showed that the characteristics symptoms caused by BBTV were small, brittle leaves with thickened veins which remained bunched at the top of the pseudostem. Plants with early infection did not produce fruits, where plants with later infection produce bunch with reduced size, weight and mishapen fingers. The characteristic symptoms caused by BBrMV were reddish spindle shaped lesion in the pseudostem, flag leaf sheath, leaf petiole, and bract. Leaves of infected plants showed characteristic chlorotic spindle shaped lesion on the leaf lamina. The characteristic symptoms of BSV were chlorotic streaks in the leaf lamina. Later the chlorotic streaks became necrotic. The characteristic symptom of CMV was mosaic pattern in the leaf lamina. The pathophysiological studies conducted in cultivar Nendran revealed that there was significant difference in carbohydrate, chlorophyll, protein and phenol content in infected plant when compared to healthy ones .. The activity of defence related enzymes like peroxidase, polyphenol oxidase and phenylalanine ammonialyase were found to be more in infected plants. Electrophoretic analysis of protein in virus infected samples through SDS-PAGE revealed the presence of an additional protein in the protein profile. The protein profile of BBTV infected sample showed one extra band with molecular weight of 20 kDa, BBrMV infected sample showed three additional protein band with molecular weight of 38 kDa, 29 kDa and 22 kDa, BSV infected sample showed three additional proteins with molecular weight of 25 kDa, 19 kDa, and 12 kDa, CMV infected sample showed one extra band with molecular weight of 25 kDa. Electrophoretic analysis of isozyme though native gel revealed the increased action of peroxidase enzyme in infected sample. Detection of VIruS infecting banana was carried out using varIOUS immunological techniques such as DAC-ELISA and DIBA using polyclonal antiserum (Agdia) and monoclonal antiserum. Both the techniques were found to be efficient in detecting virus infecting banana. Molecular diagnosis of the BBTV was carried out using CP gene and replicase gene specific primers. PCR product with amplicon size of about 530 bp was observed for coat protein gene specific primer where 237 bp was observed for replicase gene specific primer. Molecular diagnosis of BSV was carried out using two CP gene specific primers resulted in PCR product with amplicon size of 664 bp and 730 bp. Molecular diagnosis of CMV was canied out using CP gene specific primer resulted in PCR product with an amplicon size of 687 bp. CP gene specific primer for BBrMV did not give positive result. Cluster dendrogram analysis revealed that the BBTV isolate was mostly related to BBTV coat protein gene of Burundi isolate, BSV isolate was mostly related to banana streak virus isolate Trichi, CMV isolate was mostly related to cucumber mosaic virus isolate Trichi coat protein gene. The meristematic region of the virus infected banana suckers were excised and inoculated to MS media with BAP and NAA. The regeneration of plants from meristematic region was difficult because of high phenol production and contamination by endogenous bacteria. Meristem culture eliminated BBTV, CMV and BBrMV but not the BSV. Based on the research result, the banana VIruses can be detected usmg immunological and molecular technique and the meristem culture can eliminate all the banana viruses except BSV.ThesisItem Open Access Biorational management of major pests in brinjal (Solanum Melongena L.)(Department of Agricultural Entomology, College of Agriculture, Padannakkad, 2014) Gowrish, K R; KAU; Ramesha, BBrinjal (Solanum melongena L.) is a plant of considerable conomic importance in many tropical and sub tropical parts of the world. It is a popular vegetable grown as poor man’s crop in India. It is a native of India and is grown throughout the country. The brinjal crop is usually infested by a number of pests right from nursery stage till harvest. Among the insect pests, the most important and destructive ones are shoot and fruit borer, Leucinodes orbonalis; epilachna beetle, Epilachna vigintioctopunctata; leaf roller, Antoba olivacea and hairy caterpillar, Selepa docilis. For the management of these pests, the conventional pest management practices often provide unsatisfactory results. Further, the use of chemical pesticide results in insecticide resistance, pest resurgence, destruction of natural enemies and pesticide residue in the harvested produce and related environment and health hazards. Hence, there is an impetus for research and development of eco-friendly and biorational alternative for the sustainable management of major pests of brinjal. A survey was conducted in the three agro climatic regions of Kasargod district viz., low, mid and high land area. The survey revealed that per cent incidence of pests like L. orbonalis, E. vigintioctopunctata and A. olivacea were severe on brinjal in low, mid and high land area of Kasargod district. However, severe infestation of S. docilis was noticed in low land, compared to mid and high land area of Kasargod district. Spinosad 45 SC was found to be effective in controlling the shoot and fruit borer of brinjal during both summer and kharif season. Azadirachtin 1% was found to be most effective in controlling E. vigintioctopunctata adults as well as grubs. It was also found to be effective in reducing mean per cent of leaf infestation during both summer and kharif season. The Bt formulation was found to be highly effective for early instar larvae of A. olivacea. However, for older instar caterpillars, malathion 50 EC was found to be effective during both summer and kharif season. The azadirachtin 1% was found to be highly effective against S. docilis as compared to other treatments during both summer and kharif season. The spinosad 45 SC was safer to natural enemies like predatory coccinellids and spiders in brinjal ecosystem. The yield of brinjal plant was higher in spinosad 45 SC treated plots and a higher return was obtained worth Rs. 2.52 and Rs. 2.67 during both summer and kharif season respectively for every one rupee invested. Effect and compatibility of spinosad 45 SC on entomopathogenic fungi using poisoned food technique under in vitro condition revealed that spinosad 45 SC is not compatible with M. anisopliae and B. bassiana in terms of colony development, sporulation and spore germination. Residue of major and minor component of spinosad 45 SC were below the maximum residue limit.ThesisItem Open Access Innovations in e-agricultural extension technology (e-AET): diffusion and adoption of agri-expert systems among extension professionals in Kerala(Department Of Agricultural Extension, College Of Agriculture, Vellayani, 2014) Modem, Ravikishore; KAU; Allah, ThomasThe present study entitled 'Innovations in e-Agricultural Extension Technology (e-AET): Diffusion and adoption of agri-expert systems among extension professionals in Kerala' was conducted at Thiruvananthapuram district during 2012-2014 covering 100 extension professionals. Expert systems allows the use and application of information technology and communications technology (lCT's) to access and obtain information related to agricultural production, marketing, distribution, and prices, and the results of agricultural research, innovations, to raise the level of agricultural production and benefit the farming community. The present study, therefore, is with the objective to conduct a systematic appraisal of existing expert systems in agriculture vis a vis their diffusion among the extension professionals. The findings demonstrate that most of the extension professionals either in State Department, NGO or University have positive attitudes towards expert system. Age, training, innovativeness, retrievability, relevancy, format clarity, information content, availability, accuracy and timeliness affect extension professionals' attitudes. Based on respondent's stage in the adopter categorisation with reference to expert systems, it was found that 10 per cent of the sampled respondents belonged to innovators category, 19 per cent respondents belonged to early adopters' category, 32 per cent respondents belonged to early majority category, 24 per cent respondents belonged to late majority category and 15 per cent respondents belonged to laggards' category. Effectiveness index of expert system applications was worked out using seven statements and the results showed that pedagogy (as a means to effective learning through expert system) having highest effectiveness index. The findings demonstrate that most of the respondents belonged to middle age category, holding with master degrees; attended training on lCT. It was also found that most of the respondents having high innovativeness and accessibility, forinat clarity and information content of the expert system perceived as high. Availability, retrievability, relevancy, timeliness, accuracy and effectiveness index of expert system perceived as medium by the respondents. Hence, the study undoubtedly exhibited affirmative reaction from all three categories of respondents on the applications of expert systems in the field of agriculture, because local information resource centers are gaining importance with computers carrying expert systems to help fanners to make decisions.ThesisItem Open Access Physico-chemical properties of rain water harvested under different situations in lateritic(Department of soil science and agricultural chemistry, College of horticulture,Vellanikara, 2014) Ibrahim Hassen, Abdu; KAU; Betty BastinRain water harvesting is universally accepted as an important measure of water conservation throughout the world. The quality of irrigation water has become a more serious problem than quantity in different parts of the world. The characterization of quality of water is crucial for assessing the suitability for i rrigation. Hence a study was taken up on “Physico-chemical properties of rain water harvested under different situations in lateritic soil’’ in the main campus of Kerala Agricultural University, Vellanikkara during September, 2012 to August 2013. The objective of the study was to compare the physico-chemical properties of rain water from different water sources in lateritic soil. Water was collected from five sources viz, rainfall (RF), rain water harvesting pond (RWH) , Kotteppadom pond (KP), well water (W) and surface runoff (SR). The experiment for surface runoff study was laid out in an area with a gentle slope between 5 – 10 per cent . Four rain pits were dug in this area with dimensions of 0.5 m x 0.5 m x 0.5 m and lined by polyethene sheet. Water samples were taken from these water sources for one year at monthly intervals and they were analyzed for various physicochemical parameters such as colour, turbidity, pH, EC, TDS, COD, BOD, SAR, RSC, NO 3 - , Cl - and Fe. The amount and distribution of rainfall received as well as the inflow to the rain water harvesting pond were also studied. Soil samples were collected from around rain-pits before and after rains and analyzed for the content of nutrients. The total quantity of rainfall during the study period was 2872.0 mm. The maximum amount of rainfall was observed in June and the minimum in January, 2013. The amount of rain water harvested in pond during the study period was 625.48 m which comes to 63 per cent of its storage capacity. The quality of water from different sources was compared based on the results of physico-chemical analysis. It was found that pH was highest (6.69) for water from Kotteppadom pond during summer and lowest (5.54) for water from rainfall during post monsoon season. The EC and TDS values were maximum for well water during pre- monsoon season and there was significant difference among the different sources. There was no significant difference among the sources of water as regards the content of Cl and NO 3 - over the different seasons. The values for BOD and COD varied significantly over the different sources as also the seasons. Significant difference was observed for SAR and RSC values among the different sources and seasons. Loss of nutrients from soil via surface runoff from a sloppy area was studied. Soil samples were analyzed for various physico-chemical parameters such as pH, EC, OC, 3 available N, P, K, Ca, Mg, Fe, BD, PD and WHC. The different parameters were estimated both before and after receipt of rainfall. The mean values of these parameters and percent changes along with t-value were found out. During summer, after the rains, there were significant changes for the parameters like pH, OC, as well as available nutrients like K, Ca, Mg, Fe, and the percentages of decrease were 1.67, 0.67, 37.94, 25.46, 5.62 and 8.85 respectively. The water holding capacity was also decreased by 4.80 per cent. During monsoon, available nutrients like N, P, K, and WHC decreased to the extent of 23.68, 26.24, 49.32 and 7.29 per cent respectively. In general, it was found that the rainfall and rain water harvested in the water harvesting pond were superior to well water, KP pond water and surface run off water. Salinity was low for water from all the sources. Surface run off in an area with moderate slope (5-10 %) resulted in loss of nutrients like K, P, Mg and Ca. -ThesisItem Open Access Silicon and boron nutrition of rice (Oryza sativa L.) in wet land soils(Department of Soil Science and Agricultural Chemistry, College of Agriculture, Padannakkad, 2014) Sainath Nagula, KAU; Biju JosephThe experiment entitled “Silicon and boron nutrition of rice (Oryza sativa L.) in wet land soils of northern Kerala” was carried out to standardize the dose and method of application of silicon and boron to rice crop in paddy soils, to evaluate its effect on available nutrient status and yield and to study the effect of silicon in alleviating the toxicity of Fe, Mn and Al in laterite derived paddy soils. The treatments were a combination of boron source as borax (soil and foliar application) and silicon sources as calcium silicate (soil application) and potassium silicate (foliar application). Two experiments, a pot culture and a field experiment were conducted with rice variety Aishwarya as the test crop. Application of silicon and boron fertilizers improved the availability of silicon and boron in soil. Soil application of calcium silicate along with borax was superior in maintaining the available silicon and boron status of soil than foliar application of potassium silicate and borax. The study revealed that the application of silicon and boron as soil and foliar application had a synergistic effect on the availability of N, P, K, Ca, Mg, S, Zn and Cu in the soil. Application of silicon as foliar application of potassium silicate 0.5 % along with borax 0.5 % spray was effective in reducing iron toxicity in the soil while the use of calcium silicate 100 kg Si ha-1 and borax 10 kg ha-1 proved to be more effective in reducing manganese and aluminium toxicity in the soil. The content and uptake of N, P, K, Ca, Mg, and S in rice were significantly improved by the application of potassium silicate 0.5 % foliar spray along with borax 0.5 % spray 3 rounds. Foliar application of silicon and boron as potassium silicate 0.5 % spray and borax 0.5 % spray was more efficient on improving the content and uptake of silicon and boron compared to soil application of calcium silicate and borax. 141 Application of potassium silicate @ 0.5 % spray + borax 0.5 % spray 3 rounds was significantly superior with respect to yield and yield attributes of rice. In a nutshell, both the pot and field experiments clearly indicated that application of Si and B significantly improves the available nutrient status of soil, nutrient uptake, grain and straw yield of rice. Foliar application of potassium silicate and borax (0.5 % each) 3 rounds at 15 days interval significantly improved the available nutrient status of soil, yield and yield attributes of rice. It was also effective on alleviating toxicity of Fe, Mn and Al in laterite derived paddy soilsThesisItem Open Access Postharvest management practices in papaya (carica papaya L) for improving shelf life(Department of Processing Technology, College of Agriculture,Vellayani, 2014) Jayasheela, D S; KAU; Sreekala, G SThe present investigation entitled “postharvest management practices in papaya (carica papaya L) for improving shelf life” was conducted at department of processing technology, College of Agriculture, Vellayani to determine the stage of harvest maturity of papaya for local and distant market and to standardize postharvest practices for improved shelf life with minimum nutritional loss. The study was carried out in three different continuous experiments. For local market, papaya fruits harvested at ¼ yellow (144.37 DFFB – Days From Full Bloom) and ½ yellow (146.12 DFFB) stages revealed ¼ maturity as the best stage of harvest due to increased shelf life. For distant market, the fruits were harvested at one stripe yellow (142.00 DFFB) and fully mature green (139.38 DFFB) and fruits harvested at fully mature green stage had more shelf life and hence selected as best stage of harvest for distant market. Investigation on the efficacy of different sanitizing agents on surface decontamination revealed that papaya fruits harvested at ¼ yellow for local market and fully mature green fruits for distant market, washed and treated with hot water at 50% C for 20 minutes and warm sodium hypochloride at 150 ppm was effective in reducing bacterial and fungal population. The effect of waxing and ethylene absorbent was studied on papaya fruits harvested for local and distant markets, sanitized with the two best sanitising agents. For local market, fruits harvested at ¼ maturity and sanitised with hot water at 500 C for 20 minutes followed by cooling, waxing and packing with ethylene absorbent (KMnO4 – Potassium permanganate) 8.0/kg fruit as sachet in corrugated fibre board boxes recorded highest shelf life (11.00 days), lowest physiological loss in weight (1.84 per cent ), lowest loss in membrane integrity (58.18 percent leakage), lowest respiration rate (30.33 mg CO2 /kg/hr), highest total soluble solids (12.660 Brix), least microbial population and less mechanical damage. For distant market, fruits harvested at fully mature green stage sanitised with hot water at 500C for 20 minutes followed by cooling , waxing and packed in corrugated fibre board boxes with ethylene absorbent in sachet (KMnO4 8.0g/kg fruit) improved the shelf life (12.33 days) of fruits and showed lowest physiological loss in weight (2.08 per cent), lowest loss in membrane integrity (57.82 percent leakage), lowest respiration rate (35.00 mgCO2/kg/hr) higher total soluble solids (11.330 Brix), microbial population and less mechanical damage. Further studies are required for the refinement of the technology for reducing postharvest losses in papaya fruits for transport to distant market.ThesisItem Open Access Enhancement of storage life of synthetic seeds of cocoa(Theobroma Cacao L.) through germination inhibition, desiccation and low temperature treatments(Department of Tree Physiology and breeding, College of forestry,Vellanikkara, 2014) Mobin, K M; KAU; Santhoshkumar, A VThe research work entitled “Enhancement of storage life of synthetic seeds of cocoa (Theobroma cacao L.) through germination inhibition, desiccation and low temperature treatments” was carried out at Plant Tissue Culture Laboratory, College of Forestry, Vellanikkara, during the period 2012-2014. The objectives of the research work were to enhance the longevity of synthetic cocoa seeds through the application of different concentrations of chemical inhibitors/osmotica, desiccation at different RH for varying durations and low temperature treatment under refrigerated conditions and hence to prepare a storage protocol for synthetic cocoa seed. Synthetic seeds using calcium alginate encapsulation technique were prepared from embryonic axes of cocoa containing ¼ cotyledon. The experiments revealed that, presence of different chemical inhibitors in storage medium significantly influenced radicle emergence of synthetic seeds of cocoa. ABA at 10 -3 M and 10 -4 M level did not inhibit the radicle emergence of synthetic cocoa seeds and seed mortality were observed by 20 th and 25 th day respectively in both the concentrations. All synthetic seeds stored in higher concentrations of coumarin (10 -2 M) and CCC (1 M) containing ½ MS basal medium were found mortal on 10 th and 15 th day respectively. The presence of lower levels of coumarin (10 -3 M) or CCC (10 -1 M) in the storage medium helped to extend the longevity of synthetic cocoa seeds for 60 days without complete loss of viability. The combination of 10 -3 M coumarin and 10 -1 M CCC in the storage medium extended the longevity of the cultures for 67 days. Low levels of osmotica in the storage medium (100 mM sorbitol and 5% PEG) did not inhibit the germination of synthetic cocoa seeds whereas, higher levels (500 mM sorbitol and 15% PEG) had a negative influence on the cultures. Among the different osmotic treatments, 250 mM sorbitol fortified medium was found to be best as it could arrest radicle emergence in 42.97 % cultures. Longevity of more than 40 days were observed for synthetic seeds subjected to desiccation treatment: 85.3% RH (36 hours), 78.6% RH (24 hours and 36 hours) and 46.6% RH (18 hours and 24 hours). A longevity of only 15 days were obtained for synthetic seeds subjected to low temperature treatment in refrigerated conditions at 4ºC. The synthetic seeds stored in 10 M CCC and 10 -3 M coumarin added storage medium, when transferred to ½ MS basal medium after 60 days showed a maximum storage life of 98 days and 92 days respectively. The study was able to enhance the storage life of synthetic cocoa seeds by 9 days compared to a previous study by Shiran, (2012). Based on the results of the experiments conducted, a protocol for short and medium term storage of cocoa seed was developed.ThesisItem 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.
