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Open Access
Population structure and genetic diversity analysis in traditional rice landraces of Kerala
(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2022-08-19) Sowmiya S; Beena, R; KAU
The study entitled "Population structure and genetic diversity analysis in traditional rice landraces of Kerala" was undertaken during 2021-22 at the Department of Plant Biotechnology and Department of Plant Physiology, College of Agriculture, Vellayani. The study was undertaken for the genotyping of fifty rice landraces using SSR markers and population structure analysis. Fifty traditional rice landraces collected from RARS, Pattambi were raised in pots and the genomic DNA from one-month-old leaf samples were isolated by CTAB method. Fifty DNA samples were screened using thirty microsatellite primers and out of which five primers (RM431, RM154, RM85, RM5735, and RM104) did not show any amplification. Also, three SSR markers viz., RM554, RM246, and RM3825 amplified monomorphic banding patterns and hence they were not considered for further analysis. Twenty-two markers which yielded polymorphic bands were thus selected for final analysis. The Polymorphism Information Content (PIC) values for polymorphic markers ranged from 0.13 to 0.49. The primers which showed the highest PIC values were RM10793 (0.49) followed by RM310 (0.49) and RM5749 (0.48). The population structure of the fifty rice landraces performed using a software (STRUCTURE V2.3.4) divided the fifty rice landraces into four sub-populations. Phenotypic data of the same genotypes were collected from the Department of Plant Physiology, College of Agriculture, Vellayani and utilized to correlate the phenotypic data with genotypic data and to identify the tolerant landraces. Cluster analysis based phenotypic data resulted in four major clusters which was found to be similar with that of cluster produced by genotypic data. Phenotypic traits such as photosynthetic rate, transpiration rate, number of panicles per plant, and spikelet fertility per centage were used to identify the landraces that were tolerant to high temperature. According to the phenotypic data, the spikelet fertility of sub-population 1 ranged between 20-30% in high temperature condition, photosynthetic rate ranged between 15-30 µmolCO2m-2 s -1 , transpiration rate ranged between 8-15 mmolH2OCO2m-2 sec-1 , and number of panicles per plant ranged from 11-20, and this sub-population is considered to be moderately heat tolerant. The spikelet fertility of sub-population 2 ranged between 11-25% in high temperature condition, photosynthetic 117 rate ranged between 10-25 µmolCO2m-2 s -1 , transpiration rate ranged between 6-10 mmolH2OCO2m-2 sec-1 , and number of panicles per plant ranged from 7-15, and this sub-population is considered to be heat susceptible. The spikelet fertility of subpopulation 3 ranged between <25% in high temperature condition, photosynthetic rate ranged between 5-10 µmolCO2m-2 s -1 , transpiration rate ranged between 0-5 mmolH2OCO2m-2 sec-1 , and number of panicles per plant ranged from 5-10, and this sub-population is considered to be highly susceptible to heat. The spikelet fertility of sub-population 4 ranged between 11-20% in high temperature condition, photosynthetic rate ranged between 5-10 µmolCO2m-2 s -1 , transpiration rate ranged between 5-8 mmolH2OCO2m-2 sec-1 , and number of panicles per plant ranged from 5-10, and this sub-population is considered to be highly susceptible to heat. The phylogenetic tree was constructed using UPGMA (Un-weighted pair group method with arithmetic mean) using NTSYSpc cluster analysis software, and the dendrogram showed that all fifty rice landraces were divided into four major clusters. The pattern of grouping landraces into the clusters was similar to that obtained in the population structure analysis. Based on the interpretation of all the data obtained, among the fifty rice landraces; LB-2000-28-TCR-6985, Kuruva, Ittikandan, Champan, LN9937-Cherumallaram (Vatharam), Cheruvellari, Kokkan, Kurutha chitteni, Kathikannan, and Vellari were grouped as moderately tolerant to high temperature.
Open Access
Salicylic acid and Piriformospora indica induced modulation of stress resistance mechanism in Tomato (Solanum lycopersicum L.)
(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2021) Deepthi Mohan; KAU; Roy Stephen
The study entitled “Salicylic acid and Piriformospora indica induced modulation of stress resistance mechanism in tomato (Solanum lycopersicum L.).” was conducted at College of Agriculture, Vellayani, Thiruvananthapuram, during 2020 – 2021. The primary objective of this study was to analyse the salicylic acid induced NPR1 gene expression as well as Piriformospora indica induced resistance modulation to stress and its persistence and transmission through seeds. The induced resistance in plants can be classified as Systemic Acquired Resistance (SAR), mediated by the signalling molecule salicylic acid and Induced Systemic Resistance (ISR) which was induced by the presence of non-pathogenic plant growth promoting fungi or bacteria. In the present study two treatments, plant growth promoting endophytic basidiomycete fungus Piriformospora indica (biological inducer) and salicylic acid (chemical inducer) were applied on bacterial wilt resistant (Vellayani Vijay, KAU) and susceptible (Pusa Ruby, IARI) varieties of tomato and the expression rate of Non-expresser of pathogenesisrelated genes1 (NPR1) gene was evaluated using q-RTPCR. In first generation, one set of seed were inoculated with P. indica (1%) by incorporating it into the potting mixture before filling the pot-tray cavities. After confirming endomycorhizal root colonization, leaf samples were collected for analyzing NPR1 expression. Seeds from these plants were collected and progeny seedlings were maintained for analysing transgenerational expression of NPR1 gene. Second set of seeds were sown in the nursery and two weeks old seedlings were transplanted in to pots. Salicylic acid(100ppm) was sprayed at flowering stage and NPR1 gene expression was analysed on 5th day and 7th day after treatment in leaves and compared to next generation (in two weeks old seedlings) for understanding transgenerational expression. The evaluation of stress tolerance of the treated (P. indica and S.A) plants was done on progeny seedlings of Vellayani Vijay by treating it with a low density inoculum of virulent Ralstonia solanacearum (106 cells/ ml). Similarly abiotic stress tolerance evaluation was carried out by subjecting the plants to a higher temperature in polyhouse. Physiological and biochemical observations showed that application of P. indica and salicylic acid can induce resistance by regulating the enzyme activities and other physiological parameters. The resistant variety (Vellayani Vijay) gave a relative change of 7 fold and 4 fold for NPR1 expression (on 5th day and 7th day respectively) on the application of salicylic acid. The susceptible variety (Pusa ruby) showed a relative change of 6 fold and 2 fold for NPR1 expression on the application of salicylic acid. A relative change of 7 fold and 5 fold was shown respectively for first generation and second generation on treatment with salicylic acid, which confirms a transgenerational expression of the defence related gene. Similarly P.indica also showed transgenerational expression with a relative change of 5 fold and 2 fold respectively for first and second generation. P. indica treatment and exogenous application of salicylic acid can induce resistance in plants and thus induced resistance can be transferred to progenies. Transgenerational induction might be vital to the long-term survivorship of plant species on novel environments or adapt to other forms of environmental change.
Open Access
Mapping the QTL for yield traits in bitter gourd (Momordica charantia L.)
(Centre for Plant Biotechnology and Molecular Biology, College of Agriculture, Vellanikkara, 2022) Lavale, Shivaji Ajinath; KAU; Deepu, Mathew
Bitter gourd (Momordica charantia), being a rich source of phytonutrients such as carbohydrates, minerals, vitamins, and other medicinal compounds, has a great importance in healthy dietary habits. Breeders always seek to breed bitter gourd varieties for the traits such as early maturity and high yield. However, limited investigations have been made to identify the genetic loci governing yield related traits. Marker assisted selection (MAS) assures the presence of favourable alleles and fast recovery of recurrent parent genome in the cultivar under improvement. The success of MAS mainly depends on the availability of a marker-dense genetic linkage map locating quantitative trait loci (QTL) for the target traits. The present study “Mapping the QTL for yield traits in bitter gourd (Momordica charantia L.)” was carried out during October, 2018 to December, 2021 with the objective to map the quantitative trait loci and to develop chromosome-wise maps for the yield traits in bitter gourd. To develop the mapping population, high yielding bitter gourd cultivar Priyanka (Momordica charantia var. charantia) and a wild bitter gourd accession IC634896 (M. charantia var. muricata), were used as parents. A set of 450 microsatellites were screened for polymorphism using genomic DNA of parents and 47 were found polymorphic. Bitter gourd genome (GenBank acc. no. GCA_013281855.1) was scanned and new hypervariable microsatellites were identified using Genome wide Microsatellite Analysing Tool (GMATo) and named as KAUBG_n where n is a serial number. From the 75 microsatellites identified, 69 were validated through successful PCR amplification and 38 among them were polymorphic between the parents. This led to the development of a set of 85 markers polymorphic between the parents. Crosses were made between the parental lines and hybrids from the cross Priyanka × IC634896 yielded more number of fruits and total fruit produce compared to the reciprocal hybrid. An F2:3 population was developed through single seed descent method from the cross Priyanka × IC634896. A panel of 200 F2:3 plants were evaluated for twenty seven traits, including fruit-, flower-, seed-, vine-, and leaf-related traits, contributing directly or indirectly to the total yield. Wide variation was observed among the F2:3 plants for the traits studied. A group of ninety plants was selected from 200 F2:3 plants such that they represent the variation of the base population. Genomic DNA of these plants were genotyped using 85 polymorphic markers. Genotypic data from the screening of 85 markers in the mapping population were used to generate a linkage map spanning 1287.99 cM distance across eleven linkage groups (LGs) corresponding to eleven chromosomes, using IciMapping software. LG 7 (28 markers) consisted of maximum number of markers followed by LG 2 and LG 9, each having 11 markers. LG 1 had 10 markers whereas LG 3, 4 and 8 had seven markers each. LG 5, 6, 10 and 11 had only one marker each. LG 7 covered maximum map distance of 384.19 cM where LG 8 covered least map distance of 68.58 cM. The genetic map and phenotypic data were used to generate the QTL maps, using Inclusive Composite Interval Mapping (ICIM) method to locate twenty seven traits on Momordica genome. Sixty QTL, including 37 major QTL with LOD values ranging from 3.1 to 15.2, explaining 1.8 to 35.9 per cent of the phenotypic variation were identified for 24 traits, on seven chromosomes. Twenty three QTL were identified for fruit-traits with LOD values ranging from 3.1 to 7.6, explaining 5.5 to 35.9 per cent of phenotypic variation. Thirteen QTL were identified for flower-related traits with LOD value ranging from 3.1 to 15.2, explaining 7.0 to 26.0 per cent of phenotypic variation. Seven QTL each were identified for seed and leaf-related traits with LOD values ranging from 3.2 to 10.8 and 3.5 to 6.5, explaining 5.6 to 26.3 and 3.2 to 15.8 per cent of phenotypic variation, respectively. Ten QTL were identified for vine-related traits with 3.2 to 8.7 LOD values and explaining 1.8 to 17.6 per cent of phenotypic variation. Single marker analysis was performed to identify markers co-segregating with the yield contributing traits. There were 129 hits for the marker-trait association with LOD values more than 3.0, explaining 11.62 to 29.34 per cent of the phenotypic variation. Using the least and best performing F2:3 plants, markers S13, KAUBG_5 and KAUBG_11 were validated for co-segregation with fruit breadth, first pistillate flower node, and number of pistillate flowers and fruits per plant, respectively. This study gives insights into the relative locations of microsatellites and major effect QTL for yield traits in Momordica genome. QTL with shorter marker interval (qFrtL-8-1, qDPF-3-1, qDSF-3-1, qDSF-7-1, qFrtShp-8-1) can be directly used in MAS for improving yield characters. Linkage observed between microsatellites identified in this study with yield traits signifies their importance in further fine mapping as well as marker assisted selection. The linkage map constructed in this study, being the first with microsatellites from Momordica genome, paves the path for comparative and consensus map generation with other marker types. Further, fine mapping using markers within the identified QTL hotspots can lead to possible identification and cloning of genes underlying the yield traits.
Open Access
Detection of Banana bunchy top virus (BBTV) through colorimetric closed tube loop medicated isothermal amplification (Lamp)
(Department of Plant Biotechnology, College of Agriculture, Vellanikkara, 2021) Nahla Binth, T; KAU; Smita, Nair
Banana is an important food and cash crop world wide. India leads in world banana production with 31.5 million tons from 0.9 million ha area. The Banana Bunchy Top Disease caused by Banana bunchy top virus (BBTV) is considered to be the most economically destructive of the viral diseases. Primary transmission of disease is through infected planting material. Virus indexing of suckers and tissue culture plantlets is essential to ensure the availability of virus free planting materials. The available detection methods for the BBTV are ELISA and PCR. The ELISA is time consuming while PCR requires thermal cycling and the post amplification sample handling predisposing to sample cross contamination. A nucleic acid amplification method termed Loop Mediated Isothermal Amplification (LAMP) enables rapid, sensitive and specific amplification of template DNA under isothermal condition using a strand displacing polymerase like Bst DNA polymerase and 4 to 6 primers producing 109 copies of the target in less than 1 h. Combined with simple visual detection of amplicons using the Hydroxy Naphthol Blue (HNB) dye, closed tube detection can be made hence avoiding sample cross contaminations. Hence, the present study entitled “Detection of Banana bunchy top virus through colorimetric closed tube Loop Mediated Isothermal Amplification (LAMP)” was undertaken at the Center for Plant Biotechnology and Molecular Biology, College of Agriculture, Kerala Agriculture University, Thrissur, during the period from 2019 to 2021 with the objective to develop a colorimetric closed tube LAMP assay for rapid and sensitive detection of BBTV. As the initial step, samples were collected from 15 BBTV symptomatic banana plants, two symptomless plants and from plants showing symptoms of other viruses infecting banana such as Banana bract mosaic virus (BBrMV), Banana streak virus (BSV) and Cucumber mosaic virus (CMV). The samples were collected from Banana Research Station, Kannara. One sample was taken from healthy tissue culture raised plant at the Center for Plant Biotechnology and Molecular Biology, College of Agriculture, Kerala Agriculture University, Thrissur. Good quality DNA was obtained with Doyle and Doyle method followed by RNase treatment. For LAMP primer designing, coat protein gene, replicase gene and movement protein gene sequences of BBTV were selected as the target. The LAMP external and internal primers could be picked from all the targets, but loop primers were not available for replicase gene and movement protein gene sequences. Hence, six LAMP primers were designed targeting BBTV coat protein gene using the software Primer Explorer version 5.0. The concentration of different components in the LAMP reaction like dNTPs (0.8 to 1.8 mM), MgSO4 (4 to 8 mM), HNB (120 µM and 150µM) and betaine (0.8 to 1M) was optimized. The final reaction mixture contained 50 ng template DNA, 1.4 mM each dNTP, 0.8 μM each primer BBTVFIP and BBTVBIP, 0.2 μM each primer BBTVF3 and BBTVB3, 0.4 μM each primer BBTVLF and BBTVLB, 0.8 M betaine, 6 mM MgSO4, 1X Thermopol buffer with 2 mM MgSO4, 8U Bst polymerase large fragment and 120 μM HNB dye in 25 μl reaction volume. Isothermal amplification was set at 65 oC and end point detection made using 120 µM HNB dye in the reaction mixture where the BBTV positive samples showed colour change from violet to blue. The LAMP positive amplicons appeared as ladder like bands on agarose gel. Molecular characterization of LAMP amplicon was made with sequencing and restriction analysis. A restriction enzyme having cut site in between the LAMP internal primer flanking region was identified. When the LAMP amplicons were digested with the restriction enzyme Sau3AI having single cut site, fragments of expected size (62 bp and 89 bp) were obtained. The optimized LAMP assay was validated using 15 BBTV symptomatic samples, three healthy samples and samples showing symptoms of BBrMV, CMV and BSV, as observed by the agarose gel profile and the colour change with HNB dye. All the 15 BBTV symptomatic samples showed amplification in the LAMP assay while the healthy samples and the samples showing symptoms of other viruses remained unamplified. Comparison of LAMP assay with conventional PCR in the detection of BBTV was also made. Only 13 samples out of the 15 BBTV symptomatic samples tested positive in the PCR assay, thus showing higher sensitivity of the LAMP method. Moreover, the closed tube visual detection in LAMP avoids the post amplification sample handling. Since LAMP reaction can be carried out on a dry bath or water bath, it is a rapid and simple alternative to the available PCR based detection method for BBTV.
Open Access
Reverse transcription loop medicated isothermal amplification(RT-Lamp) for diagnosis of Banana bract mosaic virus in banana (Musa spp.)
(Department of Plant Biotechnology, Centre for Plant Biotechnology and Molecular Biology, College of Agriculture, Vellanikkara, 2021) Midhuna Madhu, K; KAU; Smita Nair
Banana (Musa spp.) is one of the most important and widely cultivated crops around the world. Banana is cultivated on 0.9 million hectares in India with a production of 30.8 million tonnes (NHB, 2020). Despite its importance, growers are not in a position to achieve high productivity due to the presence of various biotic and abiotic stresses. Viruses are a major concern to banana production causing up to 100 per cent yield reduction. The Banana bract mosaic virus (BBrMV), a ssRNA virus of the genus Potyvirus is reported to cause 40 per cent yield reduction in banana. The virus is transmitted through vegetative propagules and insect vectors like Pentalonia nigronervosa, Aphis gossypi and Rhopalosiphum maidis. Internal quarantine is necessary to prevent the spread of the virus. We need a platform for virus indexing, so that farmers can get good quality virus-free planting materials. The available detection methods for BBrMV are the Enzyme-Linked Immunosorbent Assay (ELISA) and Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). The ELISA is time consuming while the RT- PCR needs post PCR sample handling predisposing to sample cross contamination. Hence the study entitled “Reverse Transcription Loop Mediated Isothermal Amplification (RT-LAMP) for diagnosis of Banana bract mosaic virus in banana (Musa spp.)” was undertaken during the period from 2019 to 2021 at the Department of Plant Biotechnology, College of Agriculture (CoA), Vellanikkara, Thrissur, with an objective to develop a rapid and efficient molecular diagnostic method for BBrMV. The LAMP is an auto-cycling strand displacement isothermal DNA synthesis method using a strand displacement DNA polymerase. The RTLAMP is a specific, highly sensitive and relatively faster diagnostic method for RNA viruses. Initially, leaf samples of BBrMV symptomatic banana plants were collected from Banana Research station (BRS), Kannara, Kerala. Healthy leaf samples were collected from asymptomatic plants in the field and from tissue culture plants produced at CPBMB, CoA, Vellanikkara. Samples from banana plants showing symptoms of other viruses such Banana bunchy top virus (BBTV), Banana streak virus (BSV) and Cucumber mosaic virus (CMV) as were also collected from BRS, Kannara, to validate the results of the study. Three different methods using TRI reagent, Purelink Plant RNA reagent and RNeasy plant mini kit were tried for isolation of total RNA. Good quality RNA with intact bands could be obtained with RNeasy plant mini kit (Qiagen). For the RT-LAMP reaction, a set of six primers were designed using the software Primer Explorer version 5.0. Initially, three targets in the BBrMV genome namely, coat protein gene, replicase gene and movement protein gene were selected for picking LAMP primers. Due to unavailability of loop primers for BBrMV replicase gene and movement protein gene, six primers targeting the BBrMV coat protein gene were selected. All the primers were validated using BLASTN. Both one-step and two-step RT-LAMP assay was optimized for diagnosis of BBrMV. For two-step RT-LAMP assay, RNA was converted to cDNA and was used as template for isothermal amplification. Optimisation of the assay was done by varying the concentration of the reagents like MgSO4 (4-8 mM), dNTP (1.4-1.6 mM each), betaine (0.8-1 M) and Bst polymerase (4-8 U). The final optimised reaction mixture contained 40 ng cDNA, 1.6 mM each dNTP, 0.2 µM each primers F3 and B3, 0.8 µM each primers BrFIP and BrBIP and 0.4 µM each primers BrLF and BrLB, 1 M betaine, 4 mM MgSO4, 1x Thermopol buffer with 2 mM MgSO4, 4 U Bst polymerase large fragment and 120 µM HNB in 25 µl reaction volume. Incubation temperature at 65° C was found optimum. One step RT-LAMP is the method suited for routine diagnostics as the RNA sample can be directly used as the template for amplification. One step RT-LAMP reaction mixture contained reverse transcriptase and RNase inhibitor along with the other components of the LAMP reaction and RNA was used as the template instead of cDNA. The positive amplicons showed ladder like bands on agarose gel and showed a colour change from violet to sky blue in the presence of HNB dye in the reaction mixture. Molecular typing of RT-LAMP products was done using sequence analysis and restriction digestion. For restriction profiling, the enzyme Sau3AI having internal cut site in the RT-LAMP internal primer flanking region was used. The enzyme having single cut site produced fragments of size 100 bp and 45 bp. The RT-LAMP assay was validated using 12 BBrMV symptomatic samples, healthy samples and samples showing symptoms of other banana viruses like BBTV, BSV and CMV. All the 12 BBrMV symptomatic samples amplified in the RT-LAMP assay while no amplification was observed for healthy samples and samples showing symptoms of other viruses. Hence, in the current study, a rapid, sensitive and specific RT-LAMP based detection method for Banana bract mosaic virus was developed.
Open Access
Molecular characterization of blackeye cowpea mosaic virus causing mosaic disease in cowpea (Vigna unguiculata (L.) Wal) in Kerala
(Department of Plant Biotechnology, College of Agriculture, Vellanikkara, 2022) Tania Mathew, Pampackal; Radhika, N S
Open Access
Anticancer activity of stingless bee propolis on human cancer cells
(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2022) Hari Sagar, S K; KAU; Shanas, S
The present research work entitled “Anticancer activity of stingless bee propolis on human cancer cell line” was carried out in the Department of Plant Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram and Department of Genomic Science, Central University of Kerala during 2020-2021, with the objective to study the effect of propolis collected from two different genera of Stingless bees, viz., Lisotrigona sp. and Tetragonula spp. on human cancer cell line- A549. Crude propolis samples were collected from the nest of three species of stingless bees Lisotrigona sp. (Kollam), Tetragonula calophyllae (Thiruvananthapuram) and Tetragonula travancorica (Thiruvananthapuram). Propolis samples were macerated at room temperature and extracted with 95% ethanol. MTT assay was performed and IC-50 values were calculated in-order to determine the influence of propolis on the proliferation of human cancer cell line. Five different concentrations of propolis, viz., 150µg/ml, 300µg/ml, 450µg/ml, 600µg/ml and 900µg/ml respectively, were added to A549 cells for a period of 24 hrs. Cells treated with the propolis extracted from Lisotrigona sp, T. calophyllae and T. travancorica obtained IC50 values of 485.822 ug/ml, 236.6095 ug/ml and 294.223 ug/ml respectively. It was observed that, propolis treatment had an inhibitory effect on A549 cells after 24 hrs treatment in relative to the control. When the cells were treated with the right concentration of propolis, marked difference in their morphology was observed. The cells appeared to shrink and were floating.Quality and quantity of the samples were analyzed through nanodrop, which gave an absorbance value of 1.8 to 2.0 for all the samples. The concentration of the samples observed was between 30-200 ngµl-1 . The RNA samples were then subjected to cDNA conversion (iScriptTM cDNA Synthesis Kit). The cDNA of control and treated samples were then subjected qRT PCR. The real-time quantitative PCR (qPCR) was performed with the Applied Biosystems PCR system in a total volume of 10 µl and expression of different genes was studied. Gene expression analysis revealed that propolis taken from Lisotrigona sp. treated VIM was downregulated and it indicates that the cell migration and EMT formation are suppressed. propolis taken from T. calophyllae downregulated the expression of NODAL and it shows the inhibition of EMT formation. propolis taken from Lisotrigona sp. showed upregulation of CDX2 expression indicating that propolis from Lisotrigona sp. inhibits cell proliferation. 59 To conclude, the result of the study proved that, propolis obtained from the stingless bee Lisotrigona sp. showed more inhibitory effect on cancer cell line- A549 compared to Tetragonula spp.
Open Access
Editing of rice transcription factor OsMADS26 for drought tolerance through CRISPR/Cas9 system
(Department of Plant Biotechnology, College of Horticulture, Vellanikkara, 2021) Anjala, K; KAU; Rehna, Augustine
Rice (Oryza sativa L.) is the most widely consumed staple food of world’s human population belonging to Asia and Africa. Being a semi-aquatic annual plant, rice is highly prone to losses due to various environmental stresses. Many studies regarding this had revealed the need for developing varieties tolerant to abiotic and biotic stresses. Various methods like Marker Assisted Breeding, mutation breeding, RNAi, Antisense technology, ZFNs and TALENs were in use to develop elite traits for abiotic stress tolerance in crops like rice. But very recently, CRISPR/Cas9 system had come into the limelight as an efficient tool for the genetic manipulations of crops. Studies have identified OsMADS26 transcription factor as a negative regulator of drought tolerance in rice. Hence the current study, ‘Editing of rice transcription factor OsMADS26 for drought tolerance through CRISPR/Cas9 system’ was undertaken during the period from 2019 to 2021 at the Centre for Plant Biotechnology and Molecular Biology, CoA, Vellanikkara, Thrissur with an objective to develop drought tolerance in rice. The rice cultivar Nipponbare was selected for the study due to its competence in genetic transformation and regeneration. For CRISPR/Cas9 mediated targeted editing of OsMADS26 gene, guide RNAs (gRNAs) were designed using online software CRISPR-P v2. Genome sequence information of OsMADS26 gene available from rice genome annotation project was used for the study. Genomic region of OsMADS26 gene, flanking the gRNA target (~ 450 bp) was amplified using gene specific primers and sequence of the target region was confirmed using BLASTn and ClustalW analysis. The CRISPR/Cas9 binary vector pRGEB32 was used to clone the guide RNAs using BsaI restriction sites. Three gRNAs were selected for cloning based on features like on score value (higher the value better the editing efficiency), GC content, (40-60%), no. of off-target sites (Minimum number of off-target sites preferred), presence of secondary structure, location on the genome (towards 5' end of gene in exonic region is preferred) etc. The CRISPR/Cas9 construct for cloning was developed by annealing and ligating the gRNAs to the pRGEB32 vector followed by cloning in E. coli strain DH5α. The putative positive clones were identified by colony PCR and further confirmed by Sanger sequencing. The plasmids isolated from PCR positive colonies were sequenced using universal M13 Reverse primer which is present on pRGEB32 vector. The sequences of the clones were confirmed using multiple sequence alignment tool ClustalW. One colony of gRNA 1 construct (OsMADS26 #G1-1) and two colonies of gRNA 3 (OsMADS26 #G3-3 and OsMADS26 #G3-4) were found positive. The CRISPR/Cas9 constructs of OsMADS26 were then mobilized into Agrobacterium tumefaciens strain EHA105 following the Freeze-thaw method. The positive clones were identified using plasmid PCR using hygromycin gene specific primers. Positive colonies of OsMADS26 #G1-1 and OsMADS26 #G3-3 constructs in EHA105 were then used for rice genetic transformation. The seeds of Oryza sativa sub species japonica cultivar Nipponbare were inoculated into N6 medium supplemented with 3.0 mgL-1 2,4-D for callus induction. After five days, the calli were infected with Agrobacterium cultures harboring desired gRNA constructs for 1.5-2 min. Along with the gRNA constructs, an empty vector was also transformed to rice as vector control and a set of untransformed culture were also maintained. After around two days of co-cultivation, the excess Agrobacterium growth was washed-off thoroughly from the calli using the bacteriostatic agent Augmentin. The calli were then placed on selection medium containing Augmentin and Hygromycin. The hygromycin resistant calli showed proliferation after 14 days of incubation. The proliferating microcalli were then transferred to regeneration medium after 21 days. Proliferation of microcalli was observed in vector control, wild type as well as OsMADS26 #G1-1 and OsMADS26 #G3-3 co-transformed plates. The vector control and untransformed calli showed greening and shoot primordia initiation in regeneration medium. The regenerated shoots will be analyzed for mutation in future. Hence, in the current study, gRNA constructs for targeted editing of OsMADS26 gene was successfully developed and transformed in to rice cultivar Nipponbare. Rice genetic transformation suitable to our lab conditions were also optimized. Rice plants with mutations in the OsMADS26 gene is expected in future which can confer drought tolerance.
Open Access
Impact of water stress on sucrose metabolism in rice (oryza sativa L.)
(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2021) Anie, Thomas; KAU; Beena, R
The study entitled “Impact of water stress on sucrose metabolism in rice (Oryza sativa L.)” conducted at Department of Plant Biotechnology and Department of Plant Physiology, College of Agriculture, Vellayani during 2020-21. The objective was to study the impact of water stress on sucrose metabolism by analyzing the physiological and biochemical parameters and gene expression in selective drought tolerant and susceptible rice genotypes. In this study, two rice varieties, drought tolerant variety, PTB 7 (Parambuvattan) and drought susceptible, PTB 23 (Cheriya Aryan) were grown in pot culture and after the panicle initiation stage, crops were subjected to water stress by withdrawing irrigation until the plants experienced the symptoms of stress (leaf rolling). Then the various physiological parameters were studied five days after the induction of water stress. Extraction and estimation of sucrose metabolizing enzymes such as invertase, α- and β-amylase were done five days after induction of stress spectrophotometrically. Expression levels of sucrose synthase (SuSy) and sucrose transporter gene (SUC2) were analyzed from both root and leaf seven days after induction of water stress. Under water stress, physiological parameters such as cell membrane stability index, relative water content and yield trait like number of productive tillers were significantly reduced, activity of enzymes such as invertase, α- and β-amylase were increased and gene expression level of sucrose synthase (SuSy) and sucrose transporter (SUC-2), which are associated with the sucrose metabolism were upregulated. Water stress enhanced the sucrose content and reducing sugar content in rice plant. There was significant increase in root traits in PTB 7 but they were reduced in PTB 23. Also, there was a reduction in shoot biomass than the root biomass in tolerant rice variety, which leads to an increase in root to shoot (R/S) ratio. In this study, sucrose metabolism and transport were increased in both drought tolerant and susceptible variety under water stress condition. However, PTB 7 (drought tolerant) showed an improved sucrose metabolism than PTB 23 (drought susceptible) during water stress condition.

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