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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 QTL mapping for yield traits in vegetable cowpea(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2017) Ashwin Varghese, V; KAU; Deepu, MathewCowpea [Vigna unguiculata (L.) Walp.] is one of the most cultivated pulse crops in the semi-arid tropics of Asia, Africa, Southern Europe, and other parts of the world. It is used for both vegetable and fodder purpose. In India, kharif crop of vegetable cowpea is cultivated in an estimated area of 0.5 million hectares in states like Kerala, Karnataka, Tamil Nadu and Madhya Pradesh. Studies aimed at increased yield among crops were always challenged by the quantitative nature of traits. These quantitative traits are generally governed by multiple genes present in regions of the genome called quantitative trait loci (QTL). With the advent of molecular markers it is possible to localize the QTL with the help of linked markers, a process now widely known as QTL mapping. QTL mapping depicts the relative positioning of different markers on the chromosomes and their linkage to a specific trait. In cowpea, even though there has been few mapping efforts for traits such as resistance to Thrips tabaci and Frankliniella schultzei, flowering time, pod length and seed weight, an elaborate QTL map for yield and related traits is missing. Hence, the study “QTL mapping for yield traits in vegetable cowpea” was undertaken with the objective of mapping the SSR markers and identifying the quantitative trait loci for yield components in the genome of vegetable cowpea at the Centre for Plant Biotechnology and Molecular Biology (CPBMB), College of Horticulture, during February 2016 to June 2017. F3 plants maintained at CPBMB, derived from the cross of Sharika which is a pole type, long poded, high yielding but anthracnose and cowpea mosaic virus susceptible cultivar with Kanakamony which is a semi-trailing, medium-long poded, low yielding, anthracnose immune and cow pea mosaic virus resistant cultivar, were used to raise the F4 mapping population. Morphological observation for traits pod length, individual pod weight (IPW), pod number, days taken for first flowering (DTFF), total dry pod yield (TDPY), grains per pod, branch number, root length, plant height, plant weight, and response to anthracnose and cowpea mosaic virus diseases were recorded. High quality DNA was isolated from the parents and mapping population using the protocol standardized in this study. One hundred SSR primer pairs reported in cowpea were screened among the parental DNA for polymorphism. Thirty polymorphic primer sets were carried forward to genotype the F4 mapping population. The morphological and genotypic data were used to construct a linkage map using software ICIMapping. Two linkage groups, one having eight SSR markers distributed across 637 cM and another one having five SSR markers distributed across 271 cM were obtained. Two approaches, Single Marker Analysis (SMA) and Inclusive Composite Interval Mapping (ICIM) otherwise called Additive Linkage Mapping were followed for QTL mapping. LOD value threshold of 3.0 was used to determine the significance of QTL and linked markers. Multiple QTL hotspots were observed for different traits under study. An anchored marker, CLM0083 has been identified which was significantly linked to traits individual pod weight and total dry pod yield. The region between 25 cM to 125 cM on linkage group 1 had QTL hotspots harboring genes governing traits DTFF, TDPY, root length, plant length and plant height. This entire region was bracketed by two markers, CLM0244 at 24.25 cM and CLM0177 at 126.86 cM with an anchored marker CLM0008. This marker combination could be potentially used in marker assisted selection for the traits DTFF, TDPY, root length, plant length and plant height. Fine mapping of the QTL for these traits with large number of markers would provide more insights into the genes and hot spots involved in the yield contributing traits in cowpea.ThesisItem Open Access Evaluation of miRNA prediction tools and in silico analysis of micro and long non coding RNAs in sweet potato(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2018) Aswathy, M B; KAU; Sreekumar, JThe study entitled “Evaluation of miRNA prediction tools and in silico analysis of micro and long non coding RNAs in sweet potato (Ipomoea batatas L.)” was conducted at the ICAR-CTCRI, Sreekariyam. The objectives of the study is to compare different miRNA and target prediction tools and in silico analysis of the miRNAs and lncRNAs in sweet potato. The plant miRNA identification tools: NOVOMIR and miRPlant and miRNA-target prediction tools: psRNATarget and miRanda were compared. NOVOMIR and psRNATarget were found to be a better tool in miRNA identification and target prediction. MicroRNAs (miRNA) are 18-22nt small, endogenous non coding RNA that has prominent role in many biological processes. In the present study, we report the computational prediction of miRNAs and targets from expressed sequence tags (ESTs) of sweet potato. We predicted 13 novel potential miRNAs and 81 potential target genes and functionally characterized by BLASTX and BLAST2GO. The predicted target genes were credited with their role in signalling cascades, metabolism, and defence and stress responses. Another candidate that has more importance in the genome regulation is lncRNAs. lncRNAs are greater than 200 nucleotide length ncRNA candidate that holds functions at RNA level itself. RNAplonc is a plant long non coding RNA identification tool which uses 16 feature selection methods to predict long non coding RNA molecules. The present study which predicts 9215 lncRNAs and 8665 protein coding genes by RNAplonc in sweet potato for the first time using available ESTs sequences. Since there is a lack of lncRNA functional annotation tool, the functional analysis of predicted lncRNAs is quiet difficult. From the predicted miRNAs and lncRNAs two miRNAs and two lncRNAs were randomly selected for experimental validation by real time quantitative PCR using three different sweet potato varieties Sree Kanaka, ST13 and Khanjakad available at ICAR-CTCRI and compared the target gene’s expression in each variety. Validation results prove that both the miRNAs and lncRNAs shows their importance in crop improvement.ThesisItem Open Access Comparative evaluation of tools for gene regulatory network prediction and network reconstructioon using genomic data(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2018) Reshma Bhasker, T; KAU; Sreekumar, JDeveloping regulatory network of genes controlling traits which are of importance economically and commercially are gaining much significance in present times. GRN’s provide an insight into the transcriptional mechanisms that regulate the robust and stochastic gene expression and their relationship with the phenotypic variability that can be utilized for better crop improvement strategies. The former approaches for Gene Regulatory Network construction mainly rely on using gene expression data as input, but the time consumption and high cost of expression analysis paved way for developing new methodologies that make GRN prediction easier. The integration of genomic information along with gene expression data, could make the process of Gene Regulatory Network (GRN) construction more reliable than using expression data alone as input source. Using this approach, we have tried to develop the regulatory network of genes controlling immunity in cassava with special context to Bacterial blight resistance. Initially the immunity related genes in cassava were identified by protein domain search and analysis using HMMER. Cassava specific genes were further filtered for high competency, mapped and annotated to determine its biological role and function. A set of 1919 immunity related genes in cassava were identified, out of which 22 of them were specifically conferring virus resistance, 727 of them were screened for bacterial blight resistance by microarray data integration and a network was created using they predicted interactions identified from 324 genes using STRING. The networks obtained was visualized using Cytoscape and cross validated with simulated dataset generated from SynTReN. The generated network if immunity related genes in cassava could give more insight into the defence mechanism in cassava that can help in adapting better crop improvement and management strategies. A comparison of various approaches used for GRN prediction like probabilistic method, mutual information-based method, correlation-based approaches etc was also done and various tool like ARACNE, WGCNA etc were evaluated. Networks with different sizes, 50, 100 and 150 was generated and the network parameters like clustering coefficient, network density etc were compared. Clustering coefficient does not seem to vary with increase in network size but network heterogeneity and density were observed to increase. The statistical analysis of the performance of different methods resulted into a conclusion that the mutual information based approaches are better tools for Gene Regulatory Network construction than the other methods and it performed with a specificity of 75.7% and a sensitivity of 79.4%.ThesisItem Open Access Assessment of antiinflammatory and antioxidant properties of chlorophytum laxum R.Br.(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2018) Arundhathy, G B; KAU; Suja, S RThe thesis entitled ‘Assessment of antiinflammatory and antioxidant properties of Chlorophytum laxum R. Br.’ was carried out in the Ethnomedicine and Ethnopharmacology Division of Jawaharlal Nehru Tropical Botanic Garden and Research Institute (JNTBGRI), Palode, Thiruvananthapuram during the academic year 2017-2018. The objective of the study was to scientifically evaluate antiinflammatory and antioxidant properties of an ethnomedicinal plant Chlorophytum laxum R. Br. (Neeruvatti). Chlorophytum laxum R. Br. (Neeruvatti), herbaceous plant of family Liliaceae is one of the important medicinal plant seen in grasslands. The tubers of Chlorophytum laxum R. Br. were collected from the hills of Western Ghats and maintained at JNTBGRI to conduct the pharmacological studies. Extraction procedures were carried to prepare the drugs of different doses for the study. Acute oral toxicity studies in mice and antiinflammatory studies in rats, were done as pharmacological analysis. The preliminary phytochemical investigation, tubers of Chlorophytum laxum has shown the presence of secondary metabolites like carbohydrates, phenols, alkaloids, proteins, steroids, tannin, saponins and glycosides that may be responsible for its medicinal properties. The content of total phenols in the ethanolic extract of Chlorophytum laxum expressed as gallic acid equivalents per gram of dry extract is 5.12 mg GA/g of extract. Toxicity studies of tuber extract were investigated in Swiss albino mice for 14 days by the administration of 4 doses 5, 50, 300 and 2000 mg/kg body weight and no symptoms of toxicity were seen in the animals even up to the highest dose. In detailed pharmacological studies, antiinflammatory potential of tuber extract was investigated in vivo by carrageenan induced paw oedema and formalin induced paw oedema and in vitro by HRBC membrane stabilization assay. The extract were administered at doses of 50, 150 and 450 mg/kg body weight orally in adult wistar rats and the maximum percentage inhibition of paw oedema in the right hind limb was shown by ECL 450 mg/kg in both the methods. ECL at higher concentration protect significantly the hypotonicity induced haemolysis of HRBC by in vitro antiinflammatory analysis. The antioxidant effect of ethanolic extract of C. laxum showed IC50 of 36.62 μg/mL in hydroxyl radical scavenging, 68.91 μg/mL in nitric oxide radical scavenging and 135.67 μg/mL in antilipid peroxidation assay. Total antioxidant capacity of ethanolic extract of tubers of C. laxum was found to be 90.04 μg AAE/g of dry extract. The antioxidant potential of C. laxum was compared with a standard and the results obtained gives the significant effect and almost equal effect. The results of current study will help to develop a monograph of the drug for reference. These results substantiate the traditional claim of the plant for its medicinal use.ThesisItem Open Access Development of infectious clones of cassava mosaic virus and their validation(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2018) Vishnu Narayanan; KAU; Makeshkumar, TThe study entitled “Development of infectious clones of cassava mosaic virus and their validation” was conducted at the ICAR- Central Tuber Crop Research Institute (ICAR-CTCRI), Sreekariyam, Thiruvananthapuram during 2017- 2018. The major objectives of the study were cloning and characterisation of SLCMV/ICMV infected leaf samples, construction of infectious clones of SLCMV/ICMV and agroinoculation of Nicotiana benthaminana with the partial dimers constructed in order to check the infectiousness of the viral clones. The whole genome amplification of SLCMV/ICMV DNA samples were done and cloned in pUC19 vectors to obtain pSLCMV A7 (2746 bp), pSLCMV B2 (2738 bp) and pICMV A5 (2739 bp) full length clones. The sequence of pSLCMV A7 showed maximum similarity of 99 % with ‘SLCMV-[TVM1]’ sequence in NCBI blast. While the sequence of pSLCMV B2 showed maximum similarity of 99 % with ‘SLCMV-[Ker20]’ sequence in NCBI blast. The sequence of pICMV A5 showed maximum similarity of 95 % with ‘ICMV-[Mah]’ sequence in NCBI blast. In order to develop infectious clones, partial dimers were constructed for SLCMV DNA-A and SLCMV DNA-B and cloned in binary vector pPZP201. These infectious clones were successfully transformed into wild type A. tumefaciens, Ach5 strain by triparental method. Then agroinoculation of N. benthamiana with the constructed partial dimers was found to be successful. After 14 days post inoculation, plants infected with DNA-A + DNA-B partial dimers showed severe symptoms like leaf curling, stunting. The plants infected with partial dimer of DNA-A alone showed mild symptoms like upward leaf curling which confirmed its monopartite lineage. While those plants agroinoculated with partial dimer of DNA-B alone did not show any symptoms. These efficient infectious clones of cassava mosaic virus and their subsequent inoculation technique would provide a major advancement to the resistance development in cassava.ThesisItem Open Access Characterization of selected curcuma species germplasm using morphological and molecular markers(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2018) Bimal Thomas; KAU; Asha, K ICurcuma L., a perennial rhizomatous herb, is gaining global importance as a source of starch besides its medicinal property and use as a spice. Characterization of germplasm is very essential in crop plants and it is the basis for selection of accessions for use in crop improvement programmes. This research work was an attempt to characterize the fifteen selected accessions in eight species of Curcuma collected from different parts of India and maintained in the field gene bank of ICAR-CTCRI using morphological and molecular markers. Two accessions in each of C. amada, C. angustifolia, C. aromatica, C. decipiens, C. malabarica, C. raktakanta, C. zedoaria and one of C. longa were selected. These 15 accessions were morphologically characterized using 13 qualitative and 15 quantitative traits and a wide variability was observed. Dendrogram based on the morphological characters grouped the genotypes into four clusters. PCC analysis revealed that the accessions of the same species have shown more than 83% similarity except C. angustifolia. C. raktakanta accessions have shown a highest intra-specific similarity of 94%. C. decipiens accessions were found to be the highly variable from the most commonly exploited species C. longa while C. aromatica has shown highest similarity. PCA showed that the characters such as leaf midrib colour, rhizome flesh colour, leaf texture and aroma of rhizome have contributed mostly to the variability. Molecular characterization was done using 10 ISSR and 7 SSR markers. The total percentage polymorphism obtained by ISSR characterization was 94.31 while it was 91.11 percentage in the SSRs. C. angustifolia-1 was found to be highly variable from C. angustifolia-2 suggested the occurrence of intraspecific variability. The intra-specific similarity among C. raktakanta accessions were found to be highest than all other accession pairs. Clustering based on ISSR markers grouped the genotypes into five clusters while SSRs into six clusters. Mantel’s test showed a positive correlation between the morphological and molecular data. The results of the present study indicated that the morphological as well as the molecular tools were found to be very effective in the characterization of germplasm of Curcuma species for the developement of core collections and for further use in the crop improvement programmes.ThesisItem Open Access Computational prediction of mirnas in banana (musa spp.) and evaluation of their role in virus infection(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2018) Kokila Sajeev, Anurag Mathew; KAU; Sony, K BThe study entitled "Computational prediction of miRNAs in banana (Musa spp.) and evaluation of their role in virus infection" was conducted at the Department of Plant Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram and Central Tuber Crops Research Institute (ICAR-CTCRI), Sreekariyam, Thiruvananthapuram during 2016–2018. The objective of the study was to predict miRNAs in banana using bioinformatics tools and to validate and analyze their expression during BBrMV infection. Computational miRNA prediction tool NovoMIR was used for the prediction of miRNAs in banana genome. Analysis was performed with all the gene coding or nucleotide sequences of banana and 85 pre-miRNAs were predicted from 11 chromosomes. Most of the pre-miRNAs ranged from 140–180 nt. G+C% content of pre-miRNAs ranged from 24-77% and A+U% content ranged from 23-76%. MFE of pre-miRNAs were found by using an online application RNAfold web server. MFE of pre-miRNAs ranged from -20 Kcal/mol to -194.4 Kcal/mol. AMFE of pre-miRNAs ranged from -18.9 Kcal/mol to -85.4 Kcal/mol. All the predicted pre-miRNAs by NOVOMIR had MFE ≤ -20 Kcal/mol. MFEI of pre-miRNAs ranged from 0.85 to 1.82. BLAST analysis of 85 pre-miRNAs against annotated mature miRNAs in miRBase resulted in 52 mature miRNAs. The targets for the 52 mature miRNAs were predicted using the web tool psRNATarget and were functionally annotated by Blast2Go analysis server. Targets were identified for 40 miRNAs in banana genome. A total of 124 targets were found, with each miRNA having more than one target. Validation of the predicted miRNAs were done in in vitro banana plants of variety Nendran. Three months old tissue culture plants were infected with BBrMV virus by using aphids. Twenty healthy aphids were released on BBrMV infected sucker for 30 min for acquisition feeding, after starving for 10 min. These aphids were further released on tissue culture plants for 12-14 h for infection. The infection process was repeated twice a day for 7 days. For experimental validation, five miRNAs and their target genes having role in biological process were selected and stem-loop/gene specific primers were designed. RNA was isolated from healthy and infected leaf samples and reverse transcribed to cDNA. Expression analysis using RT-qPCR showed the presence of all the five miRNAs in healthy and BBrMV infected leaf samples. Out of them, miR-3900-5p, miR-9112 and miR-5417 were found up-regulated, miR-6928-5p downregulated and miR-3900-5p remain unchanged in infected samples and there was a positive correlation with the expression of their corresponding target genes. In the present study, 52 mature miRNAs have been predicted using bioinformatics tools in banana genome. Targets for 40 miRNAs were identified in banana genome. The five miRNAs selected were validated along with their targets. Expression analysis showed regulation of four of them during virus infection, indicating the possibility of their role in stress response in banana. The remaining miRNAs predicted need validation.ThesisItem Open Access Genetic diversity analysis of sweet potato (ipomoea batatas (L.) lam.) germplasm using morphological and ISSR markers(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2018) Sabarinath, V B; KAU; Shirly Raichal, AnilCharacterization of crop germplasm based on determination of amount and distribution of crop genetic diversity is necessary for proper utilization andconservation. This could be achieved through both morphological and molecular tools. This study entitled “Genetic diversity analysis of sweet potato (Ipomoea batatas (L.) Lam.) germplasm using morphological and ISSR markers” was carried out in the Division of Crop Improvement, ICAR-Central Tuber Crops Research Institute, Sreekariyam, Thiruvananthapuram during 2017-2018 with an objective to identify genetic diversity in the sweet potato germplasm based on morphological and molecular markers. ICAR-CTCRI is the National Active germplasm site (NAGS) of tropical tuber crops which maintains 1400 accessions of sweet potato at Sreekariyam and its regional Centre at Bhubaneswar. 54 accessions of sweet potato including 52 accessions from eastern states of India and two wild species I. triloba and I. aquatica were selected from this collection. The study consisted of two parts -morphological and molecular characterization. Morphological analysis was performed by using eighteen sweet potato descriptors as provided by IPGRI (CIP et al., 1991). The recorded data was analyzed statistically by various tools such as PCA and cluster dendrogram using Multivariate statistical package (MVSP 3.22). The dendrogram separated into the accessions into two principal clusters and one outlier at a Euclidean distance of 1.2. The PCA analysis revealed predominant vine colour, leaf lobes type as the major variables that contributed to the clustering of the sweet potato accessions. Molecular analysis was performed using ISSR markers. The genomic DNA was isolated from young leaves using Dellaporta et al. (1983) method. 11 ISSR primers were used for screening of fifty four accessions. After the final PCR using selected primers, the product was resolved in 2% agarose and polymorphic bands were obtained. Primers showed 89.8% polymorphism and the number of bands ranged from 5 to 16 with a mean value of 7.3 polymorphic bands per primer. A total 63 of 80 polymorphic bands were obtained. The data analysed using NTSYS PC 2.02 program generated a dendrogram, which grouped the accessions based on Jaccard‟s similarity coefficient which separated the fifty four accessions into three principal clusters. The first principal cluster comprised of 37 accessions which were grouped into many subclusters and there was lot of intra-clusteral variation. The second principal cluster consisted of 15 accessions and this principal cluster comprised of two accessions with 89% similarity which were also found similar in morphological characterization. The third principal cluster comprised of the two wild species, Ipomoea troloba and Ipomoea aquatica. The similarity between the different accessions ranged between 37-89%. The accessions S1574 and S1576 were 89% similar. The least similar accessions were S1408 and S1572, S1527 and S1572 (37%). A high diversity of 63% existed within the selected accessions.Mantel‟s test also showed significant correlation (r = 0.0985; p = 0.0003) between the molecular and morphological distance matrices. The hexaploid nature of the crop, self-incompatibility, along with the out crossing nature together might have contributed to the high variation observed among the accessions.ThesisItem Open Access Establishment of in vitro regeneration systems from callus and protoplast in capsicum frutescens L.(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2018) Jancy, J Sathyaraj; KAU; Deepa, S NairThe present study entitled “Establishment of in vitro regeneration systems from callus and protoplast in Capsicum frutescens L. was carried out in the Department of Plant Biotechnology, College of Agriculture, Vellayani during 2015-2017. The objective of the study was to establish callus culture from different explants in C. frutescens, to establish protocol for protoplast isolation from callus/leaf mesophyll and to culture protoplast. The study was carried out in two phases viz., establishment of callus culture and organogenesis; and standardization of protocol for protoplast culture. Callus was induced from leaves and internodal segments from in vitro raised seedlings. Among the 44 treatments in MS medium with different combinations of auxins (NAA, IAA, IBA, 2,4-D and picloram) and cytokinins (BA and Kn), 100 per cent callus induction was obtained in MS media supplemented with picloram 0.50, 1.00, 1.50 and 2.00 mg L-1 (C41, C42, C43, and C44), NAA 1.50 mg L-1, NAA 2.00 mg L-1(C3, C4) and BA 3.00 mg L-1 + NAA 1.00 mg L-1 (C29). Among the 78 treatments tried for organogenesis, calli obtained from C29 treatment showed organogenesis in MS + BA 3.00 mg L-1 + IBA 1.00 mg L-1 (R37) and (MS + BA 5.00 mg L-1 + IAA 2.00 mg L-1 (R61) in 41 and 90 days, respectively. The microshoots obtained recorded 83.33 per cent rooting in MS medium supplemented with IAA 1.50 mg L-1(Rt7) in 12.83 days. Leaves excised from in vitro seedlings, and calli produced in MS + BA 3.00 mg L-1 + NAA 1.00 mg L-1 (C29), were used as explants for protoplast isolation. Leaf bits incubated in cell protoplast washing (CPW) solution containing cellulase 2.00 per cent, macerozyme 0.50 per cent and mannitol 0.50 M (maintained at pH 5.8) for 6 h (DM28) in dark at 27oC, yielded (124 x 105) protoplast per g, with a viability of 95.16 per cent. The callus yielded maximum protoplast (36 x 105 protoplasts per g) in an enzyme combination of cellulase 2.00 per cent + macerozyme 0.50 per cent + mannitol 0.60 M (maintained at pH 5.8) after 4 h 91 (DM47) of incubation under same conditions. In protoplast purification, floatation medium with 21 per cent sucrose recorded maximum protoplast yield (30 x 105 protoplasts per g tissue and 10 x 105 protoplasts per g callus) and maximum viability (90.91 per cent and 100 per cent), from leaf derived and callus derived protoplast, respectively. The purified protoplasts with 10 x105 plating density initiated microcalli in liquid MS medium supplemented with mannitol 0.50 M, 2,4- D 0.50 mg L-1 and sucrose 30g L-1 (PCM5) in 45 days. Further development to visual colony formation of microcalli was obtained on addition of liquid MS medium supplemented with mannitol 0.40 M and sucrose 5g L-1, in 60 days from callus derived protoplast and in 70 days from leaf derived protoplast. In the study, maximum callusing response was obtained in MS medium with picloram 1.50 mg L-1. Organogenesis was obtained from the calli derived in MS medium with BA 3.00 mg L-1 and NAA 1.00 mg L-1. The shoot initiated from the calli in MS medium with BA 3.00 mg L-1 and IBA 1.00 mg L-1. The rooting of microshoots could be obtained in MS medium with IAA 1.50 mg L-1. In protoplast isolation, leaf gave higher protoplast yield and viability in CPW solution with cellulase 2.00 per cent, macerozyme 0.50 per cent and mannitol 0.50 M, incubated in dark for 6 h and callus, in CPW solution with cellulase 2.00 per cent, macerozyme 0.50 per cent and mannitol 0.60 M, incubated in dark for 4 h. The protoplasts purified in 21 per cent sucrose supplemented floatation medium and adjusted to a plating density of 10 x 105, initiated microcalli in liquid MS medium supplemented with mannitol 0.50 M, 2,4- D 0.50 mg L-1 and sucrose 30g L-1. The visual colony formation of microcalli was obtained on addition of liquid MS medium supplemented with mannitol 0.40 M and sucrose 5g L-1. In this study, a callus mediated in vitro regeneration system has been established in C. frutescens. A protocol has also been developed for protoplast isolation from leaf and calli, and its culture resulting in microcalli formation.
