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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 DNA fingerprinting of selected black pepper (piper nigrum L.) varieties.(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2011) Manjunath, Mogalayi; KAU; Nazeem, P ABlack pepper (Piper nigrum L.) famous as “Black Gold” and also known as “King of Spices” is one of the important agricultural commodities of commerce and trade in India since pre-historic period. The crop is the major source of income and employment for rural households in the predominantly pepper growing State of Kerala where more than 2.5 lakh farm families are involved in pepper cultivation. Karnataka, Tamil Nadu are the other major pepper producing States in the country. Kerala accounts for 80-90% of the total pepper production in the country. Idukki and Wynadu are the two major pepper producing districts in Kerala. Different cultivars/varieties are popular among the farmers and there phenotypic identity is not very distinct. The study entitled “DNA fingerprinting of selected black pepper (Piper nigrum L.) varieties” was carried out at the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture during the period 2009-2011. The objectives of the study were to characterize the released black pepper varieties of KAU using different molecular markers - RAPD, ISSR and SSR and to develop DNA fingerprint with which the variety could be identified and its fidelity detected. Seven black pepper varieties (Panniyur 1 to Panniyur 7) collected from Pepper Research Station, Panniyur and maintained at CPBMB, COH were used for the study. DNA extraction was done with CTAB (Rogers and Benedich, 1994) method with slight modification. The RNA contamination was completely removed through RNase treatment. Good quality DNA with UV absorbance ratio (A260/A280) 1.80 - 1.89 was used for further analysis. The PCR conditions were optimized for RAPD, ISSR and SSR assay. 30 RAPD, 34 ISSR and 29 SSR primers were screened with bulked DNA of black pepper varieties for amplification and those which gave reliable distinct banding pattern were selected for further amplification and fingerprinting. The PCR products obtained from RAPD, ISSR and SSR analysis were separated on 1.3 to 2 percent agarose gel and the amplification patterns recorded. The genomic DNA from each variety was amplified with 10 each of selected RAPD and ISSR primers and 8 SSR primer pairs. The amplification pattern was scored and depicted to develop fingerprint for each variety. The Resolving power (Rp) worked out for the different primers ranged between 7.42 to 9.42 in RAPD and 5.42 to 12.28 in ISSR analysis; indicating the capacity of the primers selected to distinguish the varieties. The Polymorphic Information Content (PIC) varied from 0.86 to 0.90 for RAPD analysis and it was between 0.80 and 0.89 in ISSR analysis indicating the variability among the genotypes. Distinct bands were used to develop DNA fingerprint of black pepper varieties Panniyur 1 to Panniyur 7 through RAPD, ISSR and SSR analysis. Sharing of amplicons developed for each primer with other varieties was also analyzed and demarcated with different colour codes in the fingerprints developed. Most of the amplicons were found shared among the varieties. However, the pattern of sharing was different and good enough to separate out the varieties. Combined DNA fingerprint of each variety with RAPD, ISSR and SSR data was also developed. The amplification pattern observed in RAPD, ISSR and SSR analysis was scored and analyzed for quantifying the variability among the varieties. The computer package NTSYS-Pc was used for cluster analysis. Maximum variability observed was 48 percent for the variety Panniyur 4. The varieties Panniyur 1 and Panniyur 3 having the same parentage indicated 76 percent similarity. The fingerprint developed was good enough to provide varietal identity and the analysis could reveal variability/relatedness among the seven varieties. Separate and combined fingerprints were developed for all the seven varieties through RAPD, ISSR and SSR analysis. The DNA fingerprints thus developed could be utilized for the variety registration and settling IPR issues.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.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 Development of scar marker for authentication of gender in kodampuli (Garcinia gummi-gutta var.gummigutta)(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2010) Shelke, Sunil Marotarao; KAU; Rajendran, P C (Guide)The diabetes and cardiovascular disease are the two serious obesity related life-style diseases, spreading at alarming rate throughout the world, especially in thickly populated third world countries in which India occupies the prime position. The fleshy fruit rind of Kodampuli (Garcinia gummi-gutta) is the richest natural source of anti-obesity metabolite hydroxyl citric acid (HCA). Which inhibit the conversion of carbohydrate to fats without affecting Kreb’s cycle through an enzyme ATP citrate lyase. Since Kodampuli is a polygamodioecious tree, it takes 8 to 12 years to identify the female trees. No significant reports are available for sex determination in Kodampuli on the basis of physiological, biochemical or molecular characters. Sex identification, lack of orthotropic shoots for grafting, prolonged seed dormancy, poor seed germination and lack of awareness of its pharmaceutical significance are hindering the extensive cultivation of this backyard companion crop in Kerala and other coastal regions of country. In the present study, an attempt was made to develop simple PCR based technique which can use for gender diagnostic in this plant. DNA samples were extracted from field grown 15 to 20 years old 25 male and 25 female trees and were bulked to 5 samples each by sex type. Earlier reported RAPD primers viz. Kit C1, Kit C8 and Kit C9 were screened but no significant polymorphism was observed. So a total of random 46 decamer primers were tested and six primers were selected for further analysis. On rescreening of the six selected primes viz. RN 5, RN 9, RN 10, RY 5, RY 18 and OPAH 12 only OPAH 12 reproduce male specific band in bulked and individual samples. Random amplified polymorphic DNA (RAPD) fragments were generated in the both bulks in order to identify markers that were polymorphic between male and female plants. A 550 base-pair (bp) male-specific DNA fragment generated with the OPAH-12 primer was identified. The polymorphic male specific band produced by OPAH 12 primer was eluted and cloned in pGEM-T vector, and transformed into E. coli JM 109 cells. Cloned cells were subjected to blue-white screening and transformed one was sent for sequencing The sequence obtained after vector screening was subjected to nucleotide blast search and ORF finder. It does not reveal any significant levels of homology and reading frame. Two pairs of SCAR primer were designed on the basis of sequence. These SCAR primers were checked for male and female samples but no polymorphic band was observed. The future line of work can be to screen the male and female genotypes with more number of primers to obtain larger base pair polymorphic band. That can used to convert this dominant marker to co-dominant one like SCAR marker. SCAR marker would be successfully employed in breeding experiments for Marker Assisted Selection.ThesisItem Open Access Morphological and molecular analysis of genetic stability in micropropagated banana (Musa spp) var. Nendran(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2012) Amar Ramesh, Kadam; KAU; Nazeem, P ABanana is an important fruit crop widely grown throughout the world. With the increasing demand and vast export potential coupled with the farmers desire to grow banana on a large area, in vitro propagated plants are becoming increasingly important as planting material for rapid multiplication of economically important commercial varieties. In vitro propagation has many advantages, such as higher rates of multiplying clean (pest and disease-free) planting material and the small amount of space required to multiply large number of plants. A major problem associated with micropropagation is the occurrence of genetic variation resulting from in vitro cultures, i.e., somaclonal variation amongst sub-clones of one parental line. The molecular basis of such variation is not well explained till date. The study entitled “Morphological and molecular analysis of genetic stability in micropropagated banana (Musa spp) var. Nendran” was carried out at the Centre for Plant Biotechnology and Molecular Biology (CPBMB), College of Horticulture, Vellanikkara, during the period from 2010 to 2012 with the objective of evaluation and characterization of the variation in tissue culture derived banana plants regenerated in different subcultures through in vitro organogenesis. Micropropagation of banana was carried out using male bud and sucker as explant source. Adventitious shoots were induced from both the sucker derived and male bud derived explants. The culture establishment rate and multiplication frequency were significantly low in male bud derived explants. The plantlets derived in specific subcultures (3, 6, 8, 10, 12, 14 and 16) were planted out and subjected for further evaluation. Considering vegetative characters during the field performance; plants derived from later subcultures were found inferior to others in both male bud and sucker derived plants. The fruit characters of the male bud derived plants were comparable to the mother plant though bunch weight and size was less in later subculture (10th). The CTAB procedure reported by Rogers and Benedich (1994) for the extraction of nucleic acids was used for the isolation of genomic DNA from tissue culture derived banana plants. The young unfurled leaves from healthy plants were collected early in the morning and used for the genomic DNA isolation. The RNA contamination was completely removed through RNase treatment. Good quality DNA with UV absorbance ratio (A260/A280) 1.80 - 1.89 was used for further analysis. The PCR conditions were optimized for Inter Simple Sequence Repeats (ISSR) assay. DNA isolated from each subculture were bulked and amplified using 10 selected primers. The amplification pattern was scored and analysed for quantifying the variation among the plants derived from different subcultures using male bud and sucker as explant. The computer package NTSYS-pc was used for cluster analysis. Plants derived from different subcultures were found grouped into two distinct clusters. Plants derived from upto 12th subculture were grouped in first cluster (<10 % variation) and those derived from 12th subculture onwards were found grouped in second cluster. The maximum genetic variation detected through ISSR assay was 15% for both male bud and sucker derived plants and the variation was relatively more in the plants derived in later subcultures (12th onwards). Changes in DNA methylation (addition of -CH3 to cytosine) has been hypothesized as an underlying mechanism of tissue culture induced variation due to the high frequency of quantitative phenotypic variation, the activation of transposable elements, heterochromatin-induced chromosome breakage events etc. An attempt was made to detect the extent of methylation pattern in micropropagated banana plants using Methylation Sensitive Amplification Polymorphism (MSAP) assay. In the study, four MSAP primer combinations were used to amplify the fragments cleaved by restriction enzymes MspI and HpaII (isoschizomers). For male bud derived plants a total of 39.4 percent methylation sites were detected while for sucker derived plants it was only 23.3 percent. Variation in methylation pattern was observed more in later subcultures (10th onwards) than initial. Percentage of internal methylation was relatively more compared to hemimethylation for both male bud and sucker derived plants. From the study, it was observed that the plants derived from initial subcultures showed lesser variation (<10 %) compared to later subcultures (12th onwards). The methylation pattern detected through MSAP assay was observed as an important factor inducing variation in tissue cultured banana. The ISSR and MSAP primers which detected polymorphism could be further utilized in quality control of tissue culture derived banana plants. Since the banana genome is sequenced recently, the information generated could be exploited to detect the hotspots in banana genome.ThesisItem Open Access Molecular characterization of geminivirus causing yellow vein mosaic in pumpkin (Cucurbita moschata Duch. Ex Poir)(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2011) Sahna Hamsa, N H; KAU; Girija, DThe study entitled “Molecular characterization of geminivirus causing yellow vein mosaic in pumpkin (Cucurbita moschata Duch. Ex poir.)” was carried out in the Molecular Biology Laboratory of Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara during the period 2009-2011. The objectives of the study included the molecular characterization of geminivirus causing yellow vein mosaic disease in pumpkin and developing a PCR based diagnostic kit. Yellow vein mosaic infected pumpkin leaf sample was collected from the field of Olericulture Department, College of Horticulture, Vellanikkara and total genomic DNA was extracted by CTAB method. Specific primers for coat protein and movement protein genes were designed based on the sequences of geminiviruses infecting vegetables, obtained from the NCBI database. PCR amplification was carried out using these primers. Amplicons of size ~900bp and ~700bp were obtained for coat protein and movement protein genes respectively. The purified PCR products were ligated in pGEMT plasmid vector and cloned. The recombinant E. coli cells were selected based on blue white screening on LB agar containing ampicillin layered with X-gal and IPTG. After confirmation of the inserts by colony PCR, the clones were sequenced. Sequences of 891bp and 702 bp were obtained for coat protein and movement protein genes respectively. Sequence analysis was carried out with standard bioinformatics tools. On blastn analysis both coat protein and movement protein sequences showed maximum similarity to Squash leaf curl China virus (SLCCV) from Coimbatore. For coat protein gene, full length ORF of 771bp was obtained and the ORF of movement protein was partial. Primers MP1F and MP1R with expected amplicon size of 1363bp were designed to get full length ORF (846bp) of movement protein gene. The technique was validated with DNA from 15 PYVM infected and 4 healthy pumpkin leaf samples collected from Palakkad, Thrissur and Malappuaram districts. The virus was detected only in the diseased samples. Hence these primers could be used in developing a molecular diagnostic tool to detect the virus. PCR amplifications were carried out in weed plants like Emilia sonchifolia, Ageratum conyzoides, Hibiscus surattensis and Synedrella nodiflora and crop plants like okra and ash gourd with yellow vein symptom to check whether these plants serve as the collateral hosts of the virus. PCR amplification was also performed in bitter gourd with distortion mosaic symptom. No amplifications were obtained in plants other than pumpkin. Using the primers PYVMV was detected in apparently healthy mature leaves of infected pumpkin. Hence, these primers could be used to detect latent infection. Sequence and phylogenetic analysis of coat protein and movement protein gene sequences showed maximum similarity to bipartite Squash leaf curl china virus (SLCCV) from Coimbatore. Hence Pumpkin yellow vein mosaic virus (PYVMV) from Kerala can be considered as a strain of SLCCV.ThesisItem Open Access Isolation and characterization of water stress activated protein kinase gene from black pepper(Piper nigrum L.) var. Kalluvally.(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2011) Bankar Ashok, Dnyandeo; KAU; Nazeem, P VWater stress is identified as one of the main constraint for enhancing the productivity in black pepper. To survive stress, plants employ a complex set of distinct signaling pathways that trigger stress-specific tolerance or avoidance in the organism as a whole. An important biochemical mechanism for regulating such pathways is reversible protein phosphorylation which is mediated by protein kinases. Gaining an understanding of the mechanisms that regulate the expression of these genes and functional annotation of their transcripts will be necessary for the genetic improvement of plants cultivated in extreme environments. Genotypic variations for drought tolerance have been reported in black pepper and the variety Kalluvally is one of the drought tolerant genotype of black pepper (Thankamani, 2003). In the previous studies at the Centre, water stress specific cDNA library of Kalluvally was constructed by suppression subtractive hybridization (SSH) (Kushwah, 2008). Several protein kinase genes were found to be up-regulated during the study. The present investigation was undertaken to obtain full length coding sequence information on the partial clones of protein kinase genes available in the cDNA library by reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The clones named PNK21 and PNK49 were selected from the library. The clones were revived on LB/ampicillin media and subjected to full length sequencing. Degenerate primers were designed based on protein kinase gene sequences of various crops, obtained from NCBI database to amplify the unknown ends of the transcripts by RT- PCR. Plants raised under greenhouse were screened for revival after water stress with different interval and frequency under open condition. The total RNA was isolated using TRI® reagent from stressed plants and subsequently cDNA was synthesized. The PCR amplification was carried out using degenerate primers designed for all three clones. Amplicons of size of ~600 bp for PNK21 clone and ~600 bp for PNK49 clone were obtained. Direct sequencing of PCR product of PNK21 clone was done. The sequence data obtained was merged and analysed using bioinformatics tools. Blastn analysis revealed ~50 per cent coverage with the cDNA sequences for protein kinase from database. So, further primers were designed to amplify the full length cDNA sequence by RNA ligase mediated-RACE. The 3’ end of the PNK21 cDNA was successfully amplified using RLM-RACE PCR with amplicon size of ~400 bp. The purified PCR product was ligated in pGEMT plasmid vector and cloned. The recombinant E. coli cells were selected based on blue white screening on LB agar containing ampicillin with X-gal and IPTG. After confirmation of the insert by colony PCR, the clones were sequenced. The finally enriched sequence was analysed using bioinformatics tools. Blastn and Blastx revealed maximum similarity with Ricinus communis APK1B protein kinase. The sequence indicated open reading frame and conserved domains for protein kinase and polyadenylation signal site TATAAA was found just upstream of the polyA tail at 3’ end when analysed with different Bioinformatics tools.
