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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, R
Coconut 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culture initiation, subculturing and embryo germination were standardised in earlier experiments (Siny, 2006; Sujatha, unpublished data, 2011).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 medium
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, Bhaskar
Total 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.
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 S
Theobroma 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.
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, Mathew
Cowpea [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.
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, J
The 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.
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 R
Curry 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.
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, P
Rice 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.
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 B
The 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.
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 Raju
The 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.

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