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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 Somatic embryogenesis from immature inflorescence of coconut (Cocos nucifera L.)(Department of Plant Biotechnology, College of Horticulture, Vellanikkara, 2019) Radhika, R; KAU; Sujatha, RCoconut palm (Cocos nucifera L.), the only species of genus Cocos, is a major crop plant which is propagated exclusively by seeds presently. Coconut is highly heterozygous, generally cross-pollinated and long lived. So the offspring differ greatly due to segregation, which can be assessed only after a very long juvenile phase. Breeding for crop improvement is thus a difficult and long-term process. So within a short period, in- vitro propagation, employing tissue culture techniques, offers a means of cloning improved planting material. So the objective of the study was to assess the in vitro response of immature inflorescence of coconut explants for induction and germination of somatic embryos by supplementing the medium with by-product of neera processing and neera. The media for culture initiation, subculturing and embryo germination were standardised in earlier experiments (Siny, 2006; Sujatha, unpublished data, 2011). In the present experiments, standardised sub-culturing medium with addition of different supplements were tested. Embryogenic calloids and somatic embryos were induced in standardized CM2 media (Y3 basal media containing a combination of 0.5 mgL-1 picloram, 1 mgL-1 NAA and 0.1 mgL-1 TDZ and 300-600 μM 2,4-D) from the immature inflorescence explant. Among the different stage and region of immature inflorescence tested in standardised CM2 medium, tip region of inflorescence (85.71%) with length 1-5 cm was found to be the best for calloid induction with low browning (4.76%). The adverse effect of exudation of polyphenols was reduced by inoculating youngest inflorescence with reduced thickness. Embryogenic calloids were sub- cultured into 10 media combinations. With four stages of explant (based on length), each stage with two types (tip or base) there were eighty combinations. The suitability of the by-product from neera processing as well as neera (CIS- Coconut Inflorescence Sap) as supplements for CM2 medium to culture the immature inflorescence of coconut was assessed and found that CM2 medium with 0.3 per cent activated charcoal, 4 per cent sucrose and 2 per cent supplement and also CM2 medium with 0.3 per cent activated charcoal, 4 per cent sucrose and 10 mlL-1 CIS was the best medium for obtaining cultures with calloid proliferation (92.75 % & 90.17 %) with minimal phenolic interference (11.58 % & 22.7%). Calloid proliferation was successfully achieved from all the media combinations. Chemical properties of neera supplement used in the culture mediumThesisItem Open Access DNA barcoding of Spider mites (Prostigmata:Tetranychidae) associated with Ornamental Plants(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2019) Jayalakshmi, Prakash; KAU; Haseena, BhaskarTotal area under floriculture in India is estimated to be 0.31 million hectares with a production of 2.08 million tonnes loose flowers and 0.82 million tonnes cut flowers in 2018-19. In Kerala, Thrissur district is considered as the centre of floriculture nursery business. Majority of nurseries in the area do not maintain their own sources of mother plants and hence import planting materials from other countries. Trade of commercial ornamentals has been recognized worldwide as an important invasion pathway for non-native pests, especially insects and mites. Though mites are reported as major pests of commercial ornamental plants from different parts of India, no systematic study has been conducted so far to document the diversity of mites associated with ornamental plants in Kerala. Considering this, the present study, ‘DNA barcoding of spider mites (Prostigmata: Tetranychidae) associated with ornamental plants’ was undertaken with the objectives to generate DNA barcodes for different species of spider mites infesting commercial ornamental plants of central Kerala and to find the genetic variability among them. The study included collection and culturing of spider mites, morphology based identification and molecular characterization of selected accessions. Purposive sampling surveys were carried out in commercial ornamental nurseries and homestead gardens of Thrissur and Ernakulam districts, covering 12 ornamental plants. Mite infested leaf samples were collected, brought to the laboratory and maintained separately as isoline cultures by assigning unique accession numbers. Morphological characterization of 26 isoline cultures revealed the occurrence of six species of spider mites under two genera viz., Tetranychus and Oligonychus. The genus Tetranychus was more diverse with five species viz., Tetranychus truncatus, T. urticae, T. okinawanus, T. neocaledonicus and T. marianae. The genus Oligonychus was represented by only one species, Oligonychus biharensis Hirst. Rose recorded the highest diversity of spider mites with five species. The mite species, T. okinawanus recorded wider host range with eight host plants viz., Adenium, rose, Gerbera, chrysanthemum, orchid, cairo morning glory, marigold and balsam. All the host plants except Adenium are new host records of T. okinawanus from India. The study recorded three new host plants for T. truncatus from India viz., rose, cock’s comb and marigold. In this study, T. marianae was recorded for the first time from Kerala. For molecular characterization, DNA was isolated and COI locus of 868 bp length was amplified using universal primer, specific to COI. Polymerase chain reaction (PCR) products of 12 accessions representing five species were sequenced and in-silico analysis was carried out. Homology analysis of sequences of 11 accessions showed 90-99 per cent similarity with sequences in NCBI database, which were in consensus with morphological identification. The sequence of the accession, Ros1vk (T. marianae) did not show significant similarity with any of the sequences in the NCBI database. In this study, COI sequence of T. marianae was submitted for the first time in GenBank. Barcode gaps among the species were examined by aligning the COI sequences using Clustal Omega tool and species-specific barcodes were identified at different nucleotide positions. Pairwise distance analysis of the sequences showed intraspecific divergence ranging from 0.00 to 0.036 and interspecific divergence ranging from 0.070-0.217. Phylogenetic analysis revealed the monophyly of T. truncatus and T. urticae and the close relationship of T. marianae with T. okinawanus. The study has shown that rose harbours many species of spider mites, indicating the need for imposing strict quarantine regulations for movement of planting materials of rose to avoid entry and invasion of mites into newer areas. The potential of T. okinawanus and T. truncatus to turn invasive in Kerala’s ecosystems is also brought out. The study establishes the reliability of COI locus as a marker for species discrimination in spider mites.ThesisItem Open Access Inheritance of molecular markers linked to vascular streak dieback disease resistance in hybrid progenies of cocoa (theobroma cacao L.)(Department of Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2019) Midhuna, M R; KAU; Minimol, J STheobroma cacao L. (also known as the chocolate tree) is a major cash crop and the costliest beverage crop. Andhra Pradesh is the leading cocoa producing state in India but Tamil Nadu ranks first with an area of 26,969 ha. Vascular Streak Dieback (VSD) caused by the fungus Ceratobasidium theobromae is a serious disease in cocoa. Since it is a vascular pathogen, chemicals have little effect on disease control. The most tenable and economic technique to tackle this disease is by evolving resistant materials. Kerala Agricultural University had initiated VSD resistant breeding since 1995. Seedlings from hybridization, exhibiting high levels of resistance were selected and field established. Nineteen hybrids, exhibiting resistance to VSD (after screening for a period of thirteen years), were selected for the present study. The progeny obtained from these hybrids by crossing it among themselves were used as plant materials for the study. Two thousand two hundred and thirty seven flowers were pollinated and seven pods were obtained. About two hundred and sixty nine seedlings were grown from the seven hybridized pods in which nursery screening for disease resistance was done. Inoculum was dispensed by keeping already infected seedlings around the experimental materials. High humidity was ensured by providing over head sprinkler system. Visual screening recorded one hundred and eighty seedlings as disease resistant, fourteen seedlings as partially resistant and seventy five seedlings as disease susceptible. Three ISSR markers (UBC 811, UBC 815 and UBC 857) and one SSR marker (mTcCIR42) linked with VSD resistance gene, identified and validated from the previous studies were used for screening of the one hundred and twenty seedlings out of which one hundred and six were resistant and fourteen were partially resistant. The polymorphic band of 950 bp, which was found to be linked with the gene conferring VSD resistance was recorded in seventy seven resistant seedlings and three partially resistant seedlings, when screened with the primer UBC 811. When screened with the primer UBC 815 (750 bp) and UBC 857 (450 bp), the polymorphic marker band which was found to be linked with VSD resistant gene from the previous studies, was present in only twenty five resistant and one partially resistant seedling and twenty one resistant and one partially resistant seedling respectively. When screened with SSR marker, the 200 bp marker band, which was tagged with the VSD resistant gene was detected in fourty six resistant and six partially resistant seedlings. The ISSR marker UBC 811 and SSR marker mTcCIR42 were found to be having comparatively good percentage of inheritance among the segregating progeny screened with a mean inheritance percentage of 71.70 per cent and 48.78 per cent respectively. Flanking sequences of the ISSR markers UBC 811, UBC 857 and SSR marker mTcCIR42 were extracted from the whole genome database of cocoa. The ORFs from the flanking sequences of UBC 811 were identified to be uncharacterized proteins by using BLASTp tool. One ORF from the upstream sequence of the UBC 857 had shown identity with beta tubulin chain. Analyzing the distance between the marker and the flanking region, it was deduced that UBC 857 is a part of beta tubulin gene. Two ORFs were identified from both the upstream and downstream flanking sequences of the SSR marker. Using BLASTp tool, it was analyzed that both the ORFs showed more than 97 per cent identity to beta tubulin gene. Analysing the spacing between the marker and the flanking sequences, it was deduced that both the ORFs are part of the same gene and the SSR marker mTcCIR42 lies within the beta tubulin gene. Tubulin beta chain belongs to the microtubular component of cytoskeletal elements which provides resistance by not allowing the fungi to penetrate the outer epidermal wall of the plants, hence protecting the plants from infection. The ISSR marker UBC 857 and the SSR marker mTcCIR42 are linked to the beta tubulin gene, which provides VSD resistance by giving resistance against penetration of the plant cell by the fungus. Nineteen seedlings were identified to be having three or more markers expressed. They can be planted in the field and can be further evaluated for yield and yield contributing characters. Sequence of the beta tubulin gene can be used for primer designing, which can be used for confirmation by screening in resistant genotypes of cocoa.ThesisItem Open Access DNA barcoding in genera benincasa and praecitrullus(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture,Vellanikkara, 2019) Priya Sonkamble, KAU; Narayanankutty, CBenincasa is a monotypic genus with only one cultivated species hispida. In India, Benincasa exhibits rich diversity. There is substantial variation in vegetative traits and fruitcharacters and it is difficult to distinguish ashgourd genotypes based on their external morphology alone. In spite of having enormous morphological variability, all members at present are accommodated into asingle species. The taxonomic classification of Praecitrullus is still uncertain. The marker and pollen morphology studieshave revealed that P. fistulosusis more related to B. hispida, than compared to other cucurbit species. DNA barcoding is a novel system designed to provide rapid, accurate and automatable species identification using short, standardized genomic regions as internal species tags. DNA barcoding is based on the characteristic variations on the sequences of identified genomic regions, which can distinguish individuals of a species. Species identification through barcoding is usually achieved by the retrieval of a short DNA sequence from a standard part of the genome (i.e. a specific gene region either from chloroplast, mitochondria or nuclear genome) and identifying the barcode gaps for each species. The barcode sequence from each unknown specimen is then compared with a library of reference barcode sequences derived from individuals of known identity. The study entitled “DNA barcoding in genera Benincasa and Praecitrullus was done at Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, with the objectives to assess genetic diversity and interspecies relationship among Benincasa and Praecitrullus genotypes and to check the species status of Benincasa using barcodes. Twenty-six ashgourd and five tinda accessions were morphologically screened in the field using the standard descriptor and basedaccessions were clustered, based on morphological characters recorded. The ashgourd accessions varied widely for their morphological traits,fruit size varied between0.191 to 12.0 kg. Thefruit shape, varied from club, spindle, cylindrical, oblong to ridged shape. The growth habit varied from long viny to short viny.Serrated, deep serrated and double serrated leaf margin was observed. For stem pubescencevariations recorded were hispid, downy, tomentose, puberulent, pilose,villous and hirsute. The cross compatibility of the ash gourd accessions were also checked to establish the species status. All the accessions of ash gourd were cross compatiblein both the directions, showing that they belong to the same species. Based on the morphological data of ashgourd accessions nine cluster were generated. One representative accession from each cluster and three representative accessions of tinda selected for the molecular studies. Total genomic was isolated and subjected to PCR assay. The matK and ITS2 gave the bands of 950 bp and 799 bp respectively, which were sufficient to identify barcode gaps.The markers were sequenced from 9 genotypes of ashgourd and 3 tinda genotypes.The BLAST analysis had shown that matK and ITS2 both loci is 99 per cent efficient for species discrimination in Benincasa and Praecitrullus. Barcode gap, a position in the sequence at which a particular nucleotide shows characteristic variation in all the members of a particular species, was identified for all the members of Benincasa and Praecitrullus species. Barcode gaps were identified for tinda, in comparison with Benincasa for both the loci. The barcodes in tinda were observed in matK locus at 47, 127 and 232bp. Similarly, for the locus ITS2, the barcode gaps in tinda were identified at 162, 207, 278, 279 and 288 bp. However, within Benincasa, there were no such characteristic barcode gaps, indicating that the null hypothesis of monotypic status of Benincasa is true. Phylogenetic analysis using Clustal Omega showed the variation within Benincasa species and Praecitrullus species.The barcodes developed in this study could be successfully used to solve the genetic relatedness of Benincasa and Praecitrullus species.ThesisItem Open Access Characterization of antioxidant fractions in curry leaf (Murraya koenigii L.) and molecular docking of selected bioactive compounds(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture,Vellanikkara, 2019) Deepak Prashant, Bhamare; KAU; Shylaja, M RCurry leaf (Murraya koenigii L.) belonging to the family Rutaceae is one of the extensively used spices in traditional Indian medicine against variety of ailments. Curry leaf is reported to possess several pharmaceutical properties such as antioxidant, anticancerous, antidiabetic, anti-inflammatory, antidiarrheal, analgesic and hepatoprotective. The therapeutic potential of curry leaf is due to several chemical constituents such as carbazole alkaloids, phenols, flavonols, tannins, terpenes, and lipids. The overproduction of reactive oxygen species (ROS) serve as the initiation point for many diseases like cancer, diabetes, arthritis and Alzheimer’s. The ROS can be scavenged by antioxidants but side effects have been reported for synthetic antioxidants like butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT). Hence, natural antioxidants are gaining scientific attention now-a-days. Though, the pharmacological potential of curry leaf is well understood, very few reports are available on the role of bioactive phytocompounds for curing diseases by interacting with target proteins. The study entitled “Characterization of antioxidant fractions in curry leaf (Murraya koenigii L.) and molecular docking of selected bioactive compounds” was undertaken with the objective to characterize antioxidant fractions in curry leaf through in vitro assays and to identify the most potent bioactive compound through LC-MS/MS and molecular docking analyses. Oleoresin was extracted from curry leaf (var. Suvasini) and further subjected to in vitro antioxidant assay. Antioxidant fractions from curry leaf were separated by silica gel column chromatography using hexane: ethyl acetate solvent system in various proportions (100:0, 80:20, 60:40, 40:60, 20:80 and 0:100) and further subjected to antioxidant assay. Sub-fractionation of the fraction exhibiting the highest antioxidant activity was done at five minutes interval and sub-fractions were also subjected to antioxidant assay. Sub-fractions exhibiting maximum antioxidant activity were analyzed by LC-MS/MS. Compounds identified through LC-MS/MS analysis were docked against eight target proteins for cancer, seven for diabetes, four for arthritis and four for Alzheimer’s. Mature leaves of curry leaf recorded higher oleoresin recovery of 9.16 per cent and possessed high antioxidant activity with a DPPH inhibition of 85.19 per cent. Fraction eluted with hexane: ethyl acetate (60:40) recorded the highest yield of extract (707.4 mg) and showed the highest antioxidant activity with 88.68 per cent inhibition of DPPH. Sub-fractionation of the fraction with the highest antioxidant activity has yielded 47 sub-fractions. Of the sub-fractions, 28th fraction showed the highest DPPH inhibition (91.51%) followed by 26th (91.08%), 34th (91.08), 38th (89.53%) and 40th (89.53%). The DPPH inhibition potential of sub-fractions 28th, 26th and 34th was similar to synthetic antioxidant BHA (91.89%). The LC-MS/MS analysis of these fractions revealed presence of 52 compounds in whole fraction (hexane: ethyl acetate 60:40), 62 in 26th sub-fraction, 51 in 28th and 34th sub-fraction, 49 in 38th sub-fraction and 45 in 40th sub-fraction. Seven compounds of curry leaf viz. alpha-aminodiphenylacetic acid, doxylamine, flucoxetine, histidinol, pheniramine, prometon and valylmethionine were found to interact with different targets for cancer. Maximum number of curry leaf phytocompounds interacted with targets for breast cancer, 17β hydroxysteroid dehydrogenase (17β HSD) and Polo-like kinase 1 (PLK1). valylmethionine inhibited 17β HSD and PLK1 with good binding energy of -66.7903 and -122.5233 kcal/mol respectively. Eight phytocompounds of curry leaf viz. alpha-aminodiphenylacetic acid, DL-2- aminooctanoic acid, doxylamine, flucoxetine, histidinol, pheniramine, prometon and valylmethionine interacted with seven different targets for diabetes. Maximum number of compounds interacted with the target fructose 1,6-bisphosphatase and valylmethionine inhibited the target with good docking score with a binding energy of -81.143 kcal/mol. Six compounds of curry leaf viz. alpha-aminodiphenylacetic acid, flucoxetine, norpropoxyphene, histidinol, pheniramine and valylmethionine interacted with four different targets of arthritis studied. Maximum number of compounds interacted with the target Nitric oxide synthase and it was inhibited by histidinol with good binding energy - 109.5131 kcal/mol. Five phytocompounds viz. alpha-aminodiphenylacetic acid, flucoxetine, norpropoxyphene, histidinol and valylmethionine interacted with four different targets for Alzheimer’s. Maximum number of compounds interacted with targets human beta- secretase 1, tau protein kinase and human butyrylcholinesterase. Good docking score was recorded for interaction of human beta-secretase 1 with histidinol. The study could bring about the potential of curry leaf as a natural antioxidant and could identify three safe phytocompounds viz. alpha-aminodiphenylacetic acid, DL-2- aminooctanoic acid and valylmethionine which could interact with targets for cancer, diabetes, arthritis and Alzheimer’s.ThesisItem Open Access Molecular and biochemical characterisation of aroma in biriyanicheera rice genotype(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture,Vellanikkara, 2019) Veerabhadraswamy, M; KAU; Sindhumole, PRice is the dietary staple food for as high as 62.8 per cent of world’s population and it contributes to 20 per cent of the total calorific intake of the people. Rice possesses many unique qualities which determine its value and preference in the market. The fragrance of rice has been one such special character that provides an extra preference to scented rice and fetches premium price in rice market. The aromatic rice cultivars are found to possess higher fragrance, when grown in relatively cooler conditions in hilly regions. Hence the production of such plant types is confined to only a few locations of India. The early maturing selection from a Kerala local landrace with short grains, named ‘Biriyanicheera’, when grown in normal tropical conditions was observed to have sufficient aroma. In this context, the present study focuses on the analysis of aroma in ‘Biriyanicheera’ rice genotype through molecular and biochemical methods. The research programme was carried out at CPBMB during 2017-2019. The seeds of aromatic rice varieties Biriyanicheera, Gandhakasala (from Palakkad, Thrissur and Ernakulam districts) and non-aromatic rice variety Triveni were used for the study. The biochemical characterisation was performed using finely powdered rice grains of all varieties through Gas Chromatography and Mass Spectroscopy (GC-MS). The GC-MS analysis revealed the presence of a diverse profile of volatiles present in the rice grains. The volatile compound Benzothiazole was detected in all the varieties, where as Ethyl acetate was the highest in Triveni, Biriyanicheera (Palakkad) and Gandhakasala (Ernakulam). The compounds, Nonanal and 2-hydroxy Benzaldehyde were found to be the major volatile compounds for aroma of Biriyanicheera in Palakkad region. Similarly, 4 nitrobenzylamine and 2-pentyl furan in Thrissur location and 4 nitrobenzylamine, 5- ethenyldihydro-5-methyl-2(3H)-Furanone, and Lincomycin in Ernakulam location were identified as the major compounds present in the aroma profile of the rice grains. The cooking quality parameters such as, Gelatinisation temperature, Amylose content and Gel consistency were evaluated for all the rice varieties. Alkali spreading test showed medium gelatinisation index for all three varieties which is optimum for the rice quality. Amylose content in rice grains was determined by treating the fine powder of rice with 0.1 N NaOH. The Biriyanicheera rice variety was found to contain 24.62 per cent of amylose content whereas, Gandhakasala and Triveni varieties showed 13.85 and 23.85 per cent respectively. Gel consistency of the rice varieties was analysed by measuring the gel length produced by boiling the rice powder. The gel length for Biriyanicheera (105mm), Gandhakasala (107.3mm) and Triveni (95mm) falls under soft category. Aroma strength detection by sensory evaluation was performed using the DUS (Distinctness, Uniformity and Stability) protocol. The Biriyanicheera variety grown in Palakkad district showed exceptionally higher aroma than the other locations. Similarly, Gandhakasala variety produced maximum aroma in Palakkad region. From the above results Palakkad district was found to influence in aroma production of aromatic varieties. Molecular characterisation was performed by amplifying the BADH2 gene present in all the three rice varieties. The amplified PCR products of genomic DNA were sequenced to analyse the presence of any mutations in BADH2 gene. The sequencing results of aromatic varieties Biriyanicheera and Gandhakasala revealed the presence of an 8 base pair mutation in exon 7 in both the varieties, whereas this mutation was absent in the non-aromatic variety Triveni. Hence it can be inferred that the aroma production in Biriyanicheera variety is due to similar mutation as that of the Basmati rice variety.ThesisItem Open Access Elicitation of phenyl propanoid production and expression profilint of acteoside biosynthetic genes in Artanema sesamoides Benth (Vathomv Arettii)(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2019) Monisha, G; KAU; Soni, K BThe study entitled “Elicitation of phenyl propanoid production and expression profiling of acteoside biosynthetic genes in Artanema sesamoides Benth (vathomvaretti)” was carried out during 2017-2019, in the Department of Plant Biotechnology, College of Agriculture, Vellayani. The objectives were to study the effect of elicitors like salicylic acid, methyl jasmonate, abscisic acid and yeast extract on phenyl propanoid glycosides production in A. sesamoides and to analyse the expression profile of key genes of acteoside biosynthesis pathway, such as PAL (phenylalanine ammonia-lyase), HCT (Shikimate O hydroxycinnamoyl transferase) and UGT (UDP glucose glucosyl transferase). Callus culture was established in MS medium supplemented with 0.5 mgL1 BA and 0.5 mgL-1 NAA using in vitro plants of A. sesamoides. Two week old callus cultures were transferred to liquid MS medium with 0.5 mgL-1 NAA and 0.5 mgL-1 BA and stabilized for 10 days. Elicitors viz., salicylic acid (SA; 40 and 100µM), methyl jasmonate (MJ; 15 and 25µM), abscisic acid (ABA; 20 and 50µM) and yeast extract (1 and 1.5 gL-1) were added to the cultures and incubated in orbital shaker (120 rpm) at 24 °C under dark condition for 48h. After elicitation, callus was harvested, dried and finely powdered. Phenyl propanoid glycosides were extracted by sequential extraction using solvents like hexane, chloroform and methanol. Methanol extracts were collected, purified and evaporated to get the residues. The total content of phenyl propanoid glycosides was maximum (4.160 mg/g dry weight) in callus treated with yeast extract (1.5 gL-1). The residues re-dissolved in HPLC grade methanol were analysed by HPLC using PDA detector at different wavelengths (197, 218, 254 & 330 nm). Six important PPGs viz., acteoside, artanemoside, isoacteoside, leucosceptoside, martynoside and plantainoside were identified from the methanol extract. The highest peak of HPLC chromatogram representing the most prominent antioxidant 87 phenyl propanoid glycoside (acteoside) in the extract appeared within the retention time of 59 min. YE (1gL-1) has significantly increased the content of all the major PPGs. The maximum content of acteoside (36.951%) and isoacteoside (22.220%) was obtained in calli treated with yeast extract (1.5 gL-1). The effect of elicitors on expression profile of acteoside biosynthesis genes such as PAL, HCT and UGT was analysed 24h and 48h after elicitation by real time PCR using SYBR® Green dye. Primers were designed using “Primer Express” software. RNA isolated from the callus after 24 and 48h of elicitation was converted to cDNA and the quality was confirmed by PCR using ACTIN gene specific primers. Cq values obtained in RT-qPCR for each gene was analysed using “qbase plus” software with ACTIN as the reference gene. Expression of PAL gene which acts in the upstream of acteoside biosynthetic pathway was found upregulated by all the elicitors. The highest elicitation (26 fold) was shown by SA (40μM), followed by yeast extract (18 fold with 1.5 gL-1), MJ (16 fold with 25μM) and ABA (11 fold with 50μM). Treatment with SA produced upregulation of PAL at 24h, while all other elicitors enhanced its expression at 48h. All the elicitors enhanced the expression of UGT at 48h of elicitation. The highest expression (42 fold) was with SA (100μM), followed by yeast extract (20 fold with 1 gL-1) and MJ (10 fold with 25μM). In this study, the two key genes, PAL and UGT, involved in the acteoside biosynthetic pathway were upregulated by all the elicitors. But only yeast extract significantly enhanced the in vitro production of six major phenyl propanoid glycosides (artanemoside, isoacteoside, leucosceptoside, martynoside and plantainoside) in A. sesamoides. The study shows a possible use of yeast extract in the in vitro production of phenyl propanoid glycosides.ThesisItem Open Access Investigaiton on extraction of starch from cassava (Manihot esculenta Crantz) stem(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2019) Hasmi Sulain, K K; KAU; Saravanan RajuThe study entitled “Investigation on extraction of starch from cassava (Manihot esculenta Crantz) stem” was carried out at the Division of Crop Utilization, ICAR- Central Tuber Crops Research Institute, Sreekariyam, Thiruvananthapuram during the year 2018- 2019. Cassava stem starch is an ideal source to increase the availability of starch without using additional land, water and fertilizers. Hence understanding the structural and functional properties of stem starch is an important aspect before substituting with root starch because there is a lack of knowledge of starch properties when compared to root starch. In this study, starch from cassava stem were extracted by four different methods such as extraction using water, extraction using chemicals such as Sodium hydroxide and sodium meta bi sulphate, microwave assisted extraction and ultra sound assisted extraction. The cassava genotypes suitable for extraction of starch from stem and functional properties of this stem starches were investigated. In the present study, the physiological characters of such as stem length, stem girth, stem fresh weight and stem dry weight and stem moisture content were measured in the selected genotypes of cassava. The results showed that stem length of cassava ranges from 100-300cm and the stem girth ranges from 2.5-8cm. The fresh weight and dry weight of cassava stem ranges from 0.4-2Kg and 0.2-0.7Kg respectively.The moisture content of cassava stem varies from 62-72% but the moisture content was less than 2% in all stem starches. The swelling volume was same for all the stem starches (15ml) and the solubility was less than 10%. Starches with high swelling volume and solubility and low gelatinization temperature has various applications in food industry. The water binding capacity varied from 46.09% to 77.50% for different cassava stem starches. The in vitro starch digestibility of cassava stem starches ranges from 0.1-0.3 g/g. The starch yield from cassava stem ranges from 17% to 30% and the starch content was found to be 38-55% on dry weight basis. The starch yield was found to be maximum for H-1687 and minimum for Quintal. 64 The peroxidase content in cassava stem starches ranges from 0.05-0.13ng/mg and the poly phenol oxidase content ranges from 10-30mg/g. Both this enzyme content was found to be lowest in Black Thailand and maximum for Sree Swarna and Quintal. It was found that extraction using ultra sound and micro wave assistance could increase the extractability of starch from cassava stem and the genotypes H1687 and H-226 was found to be high starch yielding varieties and thus it could be used for the extraction of starch from cassava stem. Cassava stem contain more than 30% of starch (dry mass), hence the wasted cassava stem starch can be utilized for both food and non-food applications. More over cassava can increase both food and fuel resources where cassava roots are for food and stems for fuel and even reduce poverty without using additional land.ThesisItem Open Access Integration of quanitative trait locus (QTL) for Tuber Colour variations with Genomic information in Sweet Potato(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2019) Reshma, T K; KAU; Sreekumar, JThe study entitled Integration of Quantitative Trait Loci (QTL) for tuber colour variations with genomic information in sweet potato (Ipomoea batatas L.) was conducted at section of extension and social sciences, ICAR-CTCRI. The main objective of the study was to identify the differentially expressed genes for various tuber colours in sweet potato using RNA sequenced data; to integrate QTL information on tuber colour with genomic information in sweet potato and to validate the identified candidate genes. Sweet potatoes are abundant in compounds of biological effects such as β-carotene, phenolic acids and anthocyanins which gives its unique flesh colours. Here, a comparative transcriptomic analysis was performed to reveal the differentially expressed genes in six sweet potato cultivars with varying flesh colours of white, orange and purple. A total of 22,534, 27,431, 22,590 differentially expressed genes were identified in the pairwise analysis of orange and white, orange and purple and purple and white libraries respectively. Among differentially expressed genes, 5472 were upregulated and 17,062 were downregulated in orange compared to white, 11,670 upregulated genes and 15,761 downregulated genes in orange compared to purple, 7,622 were upregulated and 14,968 were downregulated in purple compared to white. Functional annotation of transcripts associated with the carotenoid biosynthesis pathway revealed the genes involved in the carotenoid biosynthesis pathway. In the present study, alignment of flanking SSR markers sequences of the QTL controlling β-carotene trait was done with the sweet potato genome assembly showed the position of QTL region on the chromosome. Functional annotation of the identified chromosomal region resulted in the identification of five candidate genes for carotenoid biosynthesis from three QTLs for β-carotene. Transcriptome sequencing and fine mapping of QTL are the efficient ways for discovering novel genes involved in main pathways. The identification of agronomically important genes can be utilized for improvement of sweet potato by the introduction of the genes to commercial sweet potato cultivars and for marker assisted selection.
