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Assam Agricultural University, Jorhat
Assam Agricultural University is the first institution of its kind in the whole of North-Eastern Region of India. The main goal of this institution is to produce globally competitive human resources in farm sectorand to carry out research in both conventional and frontier areas for production optimization as well as to disseminate the generated technologies as public good for benefitting the food growers/produces and traders involved in the sector while emphasizing on sustainability, equity and overall food security at household level.
Genesis of AAU -
The embryo of the agricultural research in the state of Assam was formed as early as 1897 with the establishment of the Upper Shillong Experimental Farm (now in Meghalaya) just after about a decade of creation of the agricultural department in 1882. However, the seeds of agricultural research in today’s Assam were sown in the dawn of the twentieth century with the establishment of two Rice Experimental Stations, one at Karimganj in Barak valley in 1913 and the other at Titabor in Brahmaputra valley in 1923. Subsequent to these research stations, a number of research stations were established to conduct research on important crops, more specifically, jute, pulses, oilseeds etc. The Assam Agricultural University was established on April 1, 1969 under The Assam Agricultural University Act, 1968’ with the mandate of imparting farm education, conduct research in agriculture and allied sciences and to effectively disseminate technologies so generated. Before establishment of the University, there were altogether 17 research schemes/projects in the state under the Department of Agriculture. By July 1973, all the research projects and 10 experimental farms were transferred by the Government of Assam to the AAU which already inherited the College of Agriculture and its farm at Barbheta, Jorhat and College of Veterinary Sciences at Khanapara, Guwahati.
Subsequently, College of Community Science at Jorhat (1969), College of Fisheries at Raha (1988), Biswanath College of Agriculture at Biswanath Chariali (1988) and Lakhimpur College of Veterinary Science at Joyhing, North Lakhimpur (1988) were established. Presently, the University has three more colleges under its jurisdiction, viz., Sarat Chandra Singha College of Agriculture, Chapar, College of Horticulture, Nalbari & College of Sericulture, Titabar. Similarly, few more regional research stations at Shillongani, Diphu, Gossaigaon, Lakhimpur; and commodity research stations at Kahikuchi, Buralikson, Tinsukia, Kharua, Burnihat and Mandira were added to generate location and crop specific agricultural production packages.
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ThesisItem Open Access A study of sulphur metabolizing bacteria from tea garden soil for improving sulphur uptake in crop plants.(2023) Baruah, Manjistha; Barooah, MadhumitaSulphur, an important element for plant growth, is required for the synthesis of several essential vitamins, amino acids, initiation of enzymes, formation of glucosides, chlorophyll, and glucosinolates. Bacteria with sulphur metabolizing ability are at the center of sulphur cycling taking part in the oxidation, reduction, assimilation and dissimilation of sulphur in the entire ecosystem. In acidic soil, the amount of sulphur is less which affects the plant growth. Sulphur metabolizing bacteria with low pH tolerance can be used as bioinoculum to facilitate sulphur availability to the crop plants. Towards this end, sulphur metabolizing bacteria was isolated from acidic soils and characterized for sulphur metabolizing activity. The most efficient sulphur metabolizing, acid tolerant bacteria along with plant growth promoting activities isolated was identified as Priestia aryabhattai MBM3 as deduced from morphological, biochemical and molecular studies. The genes involved in sulphur metabolic pathway in the bacterial isolate grown in acid stress were upregulated during the lag phase indicating to the low sulphur availability during acid stress and hence higher expression of the gens. Further studies of the isolate for plant growth promotion in Brassica campestris L. variety JT-90-1 (Jeuti) was evaluated through pot experiments. The pot soils with pH 5 were supplemented with 100%RDF (T1), 50% RDF+bio-primed seeds (T2), bio-primed seeds (T3), along with an absolute control. Agronomic and physiological traits in relation to plant height, number of leaves, flavonoid content and total carbohydrate fared better in plants treated with bacteria (T2 and T3) than in control and T1 plants. At a molecular level, the plant sulphur transporters (SULTR1 and SULTR4) and pathway genes such as APS reductase and sulfite reductase had lower expression in roots indicating to favorable uptake of sulphur by the plant and its subsequent transfer to the other parts. This was evidenced by higher expression of the transporter genes in leaves signifying its uptake followed by its subsequent assimilation by the pathway genes viz. ATP sulphurylase, APS reductase, and sulfite reductase. However, in the control and T1 plants, a significant high expression of APS reductase was observed indicating over production of sulfite. Increased sulfite production is reported to affect chlorophyll content and stunted growth in plants as evidenced by agronomic and physiological traits of the control and T1 plants. The above results suggest that isolate MBM3 was able to elicit a sulphur uptake response in Brassica campestris grown in acidic soil of pH 5 as compared to control revealing a delicate symbiosis between plant and bacterial signaling pathways. Further studies to unravel the plant signaling pathways involved in evoking an enhanced sulphur metabolism due to bacterial treatment will shed more light into role of the bioinoculum before it is taken to the field.ThesisItem Open Access MOLECULAR ANALYSIS FOR TRAITS RELATED TO DROUGHT ADAPTATION IN RICE (Oryza sativa L.)(2023) BORDOLOI, SUBHROTA; Baruah, Akhil RanjanDrought is a major abiotic constraint for rice production worldwide. Model based studies revealed reproductive stage drought stress (RSDS) and intermittent drought stress (IMDS) are expected to reduce yield significantly in Sali and Ahu season, respectively. The present study was conducted with the aim to detect the genetic effects of water stress through quantitative trait locus (QTLs) associated with drought tolerance in rice. The donor and the recipient parent were selected based on the phenotypic evaluation and gene expression analysis using five drought responsive genes in two drought tolerant cultivar (ARC10372, Koimurali) and two susceptible cultivar (Disang, IR-64). Based on the results, Koimurali was crossed with a drought susceptible cultivar, Disang to obtained true F1 seeds. Subsequently the single F1 seeds was selfed for and advanced to subsequent generations (F5:6) using single seed descent (SSD) method to develop the mapping population consisting of 247 RILs. Parental polymorphism study was conducted by screening 600 SSR markers; among which 98 SSR markers were found to be polymorphic (16.33%). The RILs along with their parents were genotyped using the 98 polymorphic SSR markers and a linkage map was constructed covering a distance of 2495.31cM spanning across the 12 linkage groups using the software JoinMap 4.0. The phenotyping score of two different stress condition (reproductive stage drought stress stress and intermittent drought stress condition) were compared with the genotypic data for identification of QTLs. The QTL analysis detected eight QTLs at reproductive drought stress located on chromosome number 3(qGY3LOD 3.54, qYSI3LOD3.54); 4(qGY4LOD 2.57, qTOL4 LOD 2.56, qYSI4 LOD 2.56, qYSI4 LOD 2.59, qRYR4 LOD 2.58, qDSI4, LOD 2.62) and 11 QTLs at intermittent drought stress located on chromosome1 (qFLW1, LOD 2.23, qNOC1 LOD 2.7, qNOG1 LOD 1.83); 3(GLW3 LOD 2.55), 4(qGY4 LOD 2.7, qRYR4 LOD 2.35, qTOL4 LOD 3.5, qTOL4 LOD 2.2, qYSI4 LOD 2.35), 7(qNOG7 LOD 2.91, qTNG7 LOD 2.84). The detected QTL regions were scanned to identify putative genes associated with drought tolerance in rice in both the stress conditions. The putative gene in reproductive drought stress were used to check the expression in four extreme breeding line of different drought tolerant capacities. QTL comparision between both the stress conditions were evaluated and one QTL was found common and few shared the same chromosome but in different marker intervals indicating different genetics might control the underlying stress conditions. The putative QTLs and candidate genes identified in the present study need to be further validated, which will be helpful for the improvement of drought tolerance in rice.ThesisItem Open Access Study the viral disease complex associated with Bhut Jolokia leaf curl disease and application of Virus Induced Gene Silencing (VIGS) in model plants for virus management(2023) Saikia, Richita; Borah, Basanta KumarBhut Jolokia (Capsicum chinense Jacq) is one of the hottest chillies and an economically important crop in North-eastern India. A common problem in cultivation of Bhut Jolokia is the occurrence of leaf curl diseases that causes huge yield losses (Talukdar et al., 2015). In the present study, we attempt to identify the associated viruses and viral components in the occurrence of leaf curl disease in Bhut Jolokia. Sequencing of the PCR amplified viral targets identified multiple viruses. The results suggest that in the same isolated samples, leaf curl disease is caused by multiple DNA and RNA viruses rather than a single virus, representing two major families-Geminiviridae and bromoviridae. We also identified novel betasatellites from Bhut Jolokia infecting samples which has less than 91% pairwise identity with rest of betasatellite sequences deposited in NCBI. The betasatellites detected in Bhut Jolokia from Assam are possibly from a novel strain of a virus and might be related to Chilli leaf curl virus. In addition, understanding the plant-virus interaction mechanisms and identification of specific plant genes playing vital role in disease development could accelerate the resistance breeding process in Bhut Jolokia. Therefore, functional analysis of plant endogenous gene in conferring resistance or susceptibility against Cucumber mosaic virus (CMV) using virus induced gene silencing (VIGS) was conducted. In this study, two Nicotiana tabacum genes- Plasmodesmata located protein (PDLP) and Soybean genes regulated by cold 2 (SRC2) were silenced using the TRV-based VIGS system. The results obtained from the experimental work done suggests that both PDLP and SRC2 are potential candidate susceptibility or resistant genes against CMV infection in Nicotiana benthamiana plants. We also reported that TRV-based VIGS vectors can be used to effectively silence these potential candidate genes in Bhut Jolokia. The role of the newly identified betasatellite in promoting virulence of the cognate virus in still unclear. Thus, further complemention assay is required with or without the cognate virus in the host plant. Transgene free genome edited virus resistant plants can also be developed as a long-term goal using CRISPR technology. Further functional studies are required to validate the role of these genes in conferring resistance or susceptibility to CMV in host plants. Overall, the present study provides insight into the causal infecting viral disease complex in Bhut Jolokia and the role of plant endogenous genes in conferring resistance or susceptibility against CMV using VIGS.ThesisItem Open Access STUDIES TOWARD DEVELOPMENT OF SEED LESS BHIMKOL BANANA (Musa balbisiana) USING TISSUE CULTURE APPROACH(2023) Gogoi, Manab Bikash; Sarmah, Bidyut KumarBhimkol Musa balbisiana (Family Musaceae) under BB diploid genomic group (2n=2x=22) of banana is indigenous cultivar of Assam and has a traditional bond with the indigenous people of this region. Bhimkol is highly nutritious and possess medicinal properties. Fresh ripe pulp of the fruit has antiperoxidative and antioxidant properties which can prevent oxidative stress related diseases. Commercial importance of Bhimkol was realized with presence of industrial baby food products like Bhimvita, Bhim Shakti, Assam Bhim in the supermarket and online platforms like Amazon made from fresh fruit. The demand of ‘baby food product is increasing day by day not only in North eastern states but in other parts of India. However, presence of seed in the fruit is undesirable not only from consumption point of view but it impedes product development during industrial processing. Therefore, in the present study tissue culture methods towards development of seedless Bhimkol were studied. Male inflorescences were collected from orchards between 7-9 am to isolate anthers. Pollen viability test was conducted in bracts no 20 to 25 using 1% acetocarmine stain and highest percentage of viable pollen (78.16±15.32) was found in bract no 21. Microscopic study at 100x revealed the presence of uninucleate microspores along with dyad, triad and tetrad cells. Anthers containing uninucleate microspores were selected in the present study. In all, 300 number of anthers were inoculated in modified MS medium for callus induction. The highest frequency of friable androgenetic callus was observed after 60 days in modified MS medium supplemented with 2 mg/l of IAA and 1mg/l of 6-BAP. Calli were transferred in same medium for embryo germination. The germinated embryos were further treated with modified MS medium supplemented with IAA, BAP & CH for shoot induction. Growth regulators combination of 7 mg/l of BAP, 0.5 mg/l of IAA, 350 mg/l of CH was found to be the best for shoot induction and 12 numbers of plants were regenerated with a frequency of (4 %). Chromosome counting at metaphase stage revealed four (4) haploid n=11 with a frequency of (1.3%) and 8 diploids n=22 plants with a frequency of (2.66%). The ploidy level was reconfirmed by Flow Cytometer based on histogram florescence intensity associated with relative DNA content. For obtaining triploids endosperm culture was done using three types of explants 1) half cut seed, 2) endosperm and 3) endosperm containing zygotic embryo. Total 250 explants were inoculated in modified MS with different concentrations of growth hormones. Explants with half cut seed and endosperm degenerated after 28 days of inoculation due to blackening, whereas endosperm containing zygotic embryo produced white friable callus in modified MS mediim with 0.1 mg /l of 2, 4-D, 3mg/l of NAA and 0.5 mg kinetin. For shoot induction calli were transferred in different treatments of GR.Treatment no (T5) modified MS medium with 2 mg/l of NAA, 3 mg/l of BA produced the highest number of shoots regenerated and led to 26 numbers of plants. Chromosome counting, Flow cytometry test along with stomatal counts revealed 1 anueploid, 5 diploid and 20 triploids. Studies were also conducted to isolate protoplast from In vitro regenerated haploid and diploids tissue using reported protocol and demonstrated the regeneration of somatic hybrid through fusion of haploid and diploid protoplasts which was confirmed by histogram florescence intensity associated with relative DNA content using a Flow cytometer. The protocol for haploid, triploid and somatic hybridization could be used in future for increasing the frequency of haploid, triploid and somatic hybrids in order to develop seedless Bhimkol production.ThesisItem Open Access CLONING AND CHARACTERISATION OF cDNA ENCODING LEGUME SEED DEFENSIN AND ITS ACTIVITY AGAINST BRUCHIDS(2023) Dayma, Jyotsna; Acharjee, SumitaDefensins are small cysteine-rich peptides that play a crucial role in a plant’s innate immune system and are known for their well-established antimicrobial activity. However, their potential insecticidal properties have received limited exploration. Chen and his coworker have identified a defensin protein from mung bean (Vigna radiata) having insecticidal activity against bruchids. Our previous research confirmed the upregulation of a defensin gene in black gram due to the bruchid infestation. In the present study, sequencing of the full-length complementary DNA of the defensin gene from various legumes was performed. We found two amino acid sequence variants, PEP1 and PEP2 with 90 to 97% homology with the previously reported mung bean defensin protein, respectively. The PEP1 variant was found in black gram, pea, cowpea, and common bean, while PEP2 was found in mung bean, chickpea, and pigeon pea. The amino acid sequence alignment showed there are two amino acid substitutions in the signal peptide and three amino acid substitutions in mature protein regions of PEP1 compared to PEP2. Expression analysis of insecticidal defensin in different legumes revealed significant upregulation of the defensin gene in common beans, followed by black gram and cowpea, whereas, downregulation was observed in the case of pea, pigeon pea, mung bean, and chickpea that are susceptible to the bruchid infestation. The signal-truncated defensin protein was expressed in E. coli bacteria with an intein tag and a 32.5kD fusion protein was isolated. The cleavage of the tag through chitin affinity chromatography yielded mature peptides of 5.5 kD. Computational visualization showed that PEP1 displayed stronger interactions with the α-amylase enzyme of bruchids (Callosobruchus sp.), particularly through inter-chain hydrogen bonding when compared with PEP2. Moreover, PEP1 demonstrated higher (>14%) α-amylase inhibition activity compared to PEP2. In the insect bioassays, bruchids fed on PEP1 through an artificial diet at a concentration of 0.2% and 0.3% exhibited no adult emergence compared to PEP2. A plant expression vector was also constructed for expressing the defensin gene in the seeds of legumes. The results suggest the presence of amino acid substitutions in the defensin protein of legumes, which could be associated with the resistance to bruchid infestation. Thus, the expression of the black gram defensin gene regulated by a seed-specific promoter in grain legumes such as chickpea/ pigeon pea could provide durable resistance to bruchids.ThesisItem Open Access ESTIMATION OF GENETIC EFFECTS FOR LOW TEMPERATURE TOLERANCE IN boro RICE OF ASSAM THROUGH QTL ANALYSIS(2023) Kumari, Gracia Priya; Baruah, A.R.In recent years, boro rice cultivation in Assam has gained popularity among farmers due to its higher productivity than sali and ahu rice, and has become an alternative to flood affected areas. However, the advantages of boro rice cultivation are limited by low-temperature stress during seedling stage, leading to seedling mortality. Therefore, understanding genetic basis has become a prerequisite and hence the present study was aimed to detect the genetic effects for low temperature stress through QTL (Quantitative trait loci) analysis. The donor parent had been selected based on phenotypic evaluation and marker based genotypic variations for low temperature tolerance in 219 boro rice cultivars collected from Regional Agricultural Research Station (RARS), Karimganj, AAU. Among these cultivars, the donor parent was selected based on its ability to survive the lowest temperature of 5°C. On the other hand, the recipient parent was chosen from high yielding varieties (HYVs), showing susceptibility to low temperatures at 12oC, 8oC and 5oC. Two cultivars were selected as parents, namely PSBRC2 (donor) and Jyotiprasad (recipient), and were crossed to obtain true F1 seed. Subsequently, single F1 seed was selfed, F2 seeds were advanced to F7 generations using the SSD (single seed descent) method to develop a mapping population consisting of 188 RILs (Recombinant Inbred Lines). Parental polymorphism study was conducted using 600 microsatellite markers; among which 90 markers were found to be polymorphic (15% marker polymorphism). The RILs along with parents were then genotyped using 90 microsatellite markers. A linkage map was constructed covering 4391 cM based on 90 microsatellite markers spanning 12 linkage groups using the software Join Map 4.0. The phenotypic scores under field and controlled environment were compared with genotypic data using software, Map QTL6 and R/QTL for identification of the QTLs. The analysis detected six QTLs associated with seedling stage traits in controlled environment under cold stress, located on chromosomes 2 (qCTGRS2, LOD score 2.7; qCTSUR2, LOD score 2.3), 5(qREC5, LOD score 2.9; qSUR5, LOD score 2.7), 8(qREC8, LOD score 2.16), and 12 (qSUR12, LOD score 2.06). Additionally, one QTL was found to influence colouration at seedling stage in field condition on chromosome 1 (qCTS1, LOD score 3.24), and six QTLs were detected for yield attributing traits on chromosomes 2 (qNOT2, LOD score 2.47), 4 (qGY4, LOD score 1.93) and 9 (qNOG9, LOD score 2.27; qDFF9,LOD score 2.47; qSF9, LOD score 2.01).The detected QTL regions were scanned to identify candidate genes associated with cold tolerance in rice. A 10Mb region for each QTL was scanned and a total of 6995 genes were detected; out of which 3171 were annotated. From the annotated genes 32 genes were found to be stress related genes and from these, eleven(11) candidate genes associated with cold stress were detected (OsICE-1, ICE-1, ATG6A, OsATG6a, OsOFP4, OsOFP06, OsOFP6, OFP6, OsPUB3, OsPUB03, PUB3, P5CS2, OsP5CS2, OsALDH18B2, PYL/RCAR3, OsCTZFP8, BIP120, OsUGT90A1, OsFKBP65, OsFKBP62b). These findings hold significant promise for enhancing low temperature tolerance in boro rice, if candidates are identified and introgressed through fine mapping and further map based cloning.ThesisItem Open Access A Study on the Role of Rhizospheric Bacteria in Promoting Plant Growth and Alleviating Biotic Stress in its Host(2023) GHOSH, ALOKESH; Boro, Robin ChandraCabbage is susceptible to various fungal pathogens such as Xanthomonas campestris, Alternaria brassica, Rhizoctonia solani, etc. Various fungicides have been recommended such as chlorothalonil and tebuconazole, which are highly toxic to both soil and aquatic ecosystem and may induce tumors in mammalian cells. Bio-inputs can be the most sustainable and eco-friendly approach to mitigate plant diseases. In the present study, two rhizospheric bacterial isolates Bacillus amyloliquefaciens AG1B and B. subtilis AG2B showed antagonistic activity against Alternaria brassicicola AG1F. Previously reported endophytic bacteria, B. subtilis Scb-1 also showed similar antagonistic activity against the same pathogenic fungi. The cell-free supernatant of the bacterial isolates showed a significant reduction in fungal growth. Microscopic studies revealed deformed hyphae and conidia with bubble formation when co-cultured with the antagonist bacterial isolates. Lipopeptides are bioactive compounds that pose substantial challenges to the structural integrity of fungal cell walls and are regarded as antifungal in nature. LC-MS analysis showed that co-cultivation of B.amyloliquefaciens AG1B with A. brassicicola AG1F results in the secretion of several lipopeptide and polyketide compounds such as surfactin, iturin, etc. Similar results were also obtained when B. subtilisAG2B and B. subtilis Scb-1 were co-cultured with A. brassicicola AG1F, respectively.The complete genome of the potential bacterial isolate; B. amyloliquefaciens AG1B with a percentage inhibition of 76.47 % against A. brassicicola AG1F was reconstructed through de novo assembly, revealing a genome size of 3.89 Mbp. This genome encompasses genes associated with plant growth promotion, lipopeptides and polyketides synthesis, and genes that confirm its affiliation with rhizobacterial interactions. Differential expression analysis of key genes such srfAB, ItuA, fenF etc., involved in the synthesis of lipopeptides and polyketides during the dual culture condition showed significant transcriptional up-regulation, validating the results of metabolite profiling. Moreover, the bio-primed seeds of cabbage with the potential bacterial isolates, B. amyloliquefaciens AG1B, B. subtilis AG2B and B. subtilis Scb-1 respectively, showed better germination percentage, increase in root, and shoot length. Foliar application of the potential bacterial isolates B. amyloliquefaciensAG1B, B. subtilis AG2B and B. subtilis Scb-1 efficiently resulted in a reduction of disease severity up to 80%. Thus, multifaceted bio-inputs like B. amyloliquefaciens AG1B, B. subtilis AG2B and B. subtilis Scb-1 can be used for making bio-formulation for sustainable management of fungal diseases.ThesisItem Open Access GENE EXPRESSION STUDIES FOR LOW TEMPERATURE TOLERANCE AT THE BOOTING STAGE IN RICE(2023) Bora, Smriti Shyamolee; Baruah, Akhil RanjanRice (Oryza sativa L.) is a staple food crop for half of the world’s population and it has been grown as major cereal crop in three distinct seasons in Assam. Out of various constrains of rice production, abiotic stresses such as drought, low temperature (LT), salinity adversely affect crop growth and development. Low temperature stress at the early growth and reproductive stages in rice are critical, and the present study was conducted to screen low temperature tolerant genotypes at the booting stage (intermediate stage of reproductive phase) and study gene expression for cold responsive genes in tolerant and susceptible rice genotypes. A total of 30 rice cultivars collected from Regional Agricultural Research Station, Karimganj, AAU were subjected to low temperature stress at the booting stage in a growth chamber maintained at 12- 12.5C for seven days. The recovery and low temperature tolerance of plants after stress were evaluated based on pollen fertility, number of panicle emerged, number of seeds set and grain yield. The results revealed that the phenotypes of C-59 and IET-9097 were superior for all the traits under study whereas Costco and IET-8687 showed lowest trait values, indicating varied levels of low temperature tolerance at the booting stage. These four cultivars, C-59 and IET- 9097 as low temperature tolerant; Costco and IET-8687 as low temperature susceptible were chosen to study gene expression analysis using six cold responsive genes (OsCOLD1, OsDREB2A, OsCTB1, OsLTT1, OsAPX1, OsNAC9) through real time quantitative reverse transcriptase polymerase chain reaction (RT-qPCR). The results showed that all the genes were up-regulated in the cold tolerant cultivars, C-59 and IET-9097 and down regulation in the other two cold susceptible cultivars, Costco and IET-8687. After performing qRT-PCR analysis, correlation between the gene expression data and phenotypic observations followed by path analysis was performed to detect associations between the genes and phenotypes. Path analysis revealed that out of six genes used in the study, three genes showed direct positive effect over grain yield (OsDREB2A, OsCTB1 and OsNAC9) and three genes showed indirect effect for grain yield (OsCOLD1, OsLTT1 and OsAPX1). The study is preliminary with respect to low temperature tolerance at the booting stage using rice collection of AAU and hence, further validation is required.ThesisItem Open Access STUDY ON GENETIC FIDELITY IN TISSUE CULTURED RAISED PLANTLETS OF CITRUS LIMON (ASSAM LEMON)(2023) Das, Ratnakishore; Sen, PriyabrataCitrus lemon, a highly significant species both economically and nutritionally, is extensively cultivated in Assam. The demand for this valuable crop has led to the development of efficient methods for large-scale multiplication to meet the increasing requirements of the market. Micropropagation, an advanced tissue culture technique, offers a promising approach for the rapid production of genetically identical plants. In this study, Citrus lemon was micropropagated to assess its potential for large-scale multiplication, and the genetic fidelity of the micropropagated plantlets was evaluated using Simple Sequence Repeat (SSR), Inter-Simple Sequence Repeat (ISSR), and long Retrotransposon (LTR) markers. SSR markers are highly polymorphic, consisting of short repetitive DNA sequences, while ISSR markers target the regions between microsatellites. LTR markers, on the other hand, focus on retrotransposon dispersed throughout the genome Healthy and vigorously growing shoot-tip were excised from donor plants and placed on the medium. Within a few weeks, these shoots successfully developed into multiple plantlets through the process of organogenesis. The micropropagated plantlets were then acclimatized to the greenhouse environment, and their growth performance was monitored. To determine the genetic stability and fidelity of the micropropagated plantlets. Fifty plant samples including the mother plant and rest in vitro regenerated plantlets are used for testing the genetic fidelity. Total genomic DNA was extracted from both the mother plants and the micropropagated plantlets using standard protocols, and the SSR, ISSR, and LTR markers were employed to assess genetic variation, if any, between the two sets of samples. The results of the genetic fidelity analysis demonstrated no detectable variation between the micropropagated Citrus lemon plantlets and their mother plants. All the SSR, ISSR, and LTR markers revealed identical banding patterns, indicating the absence of any somaclonal variation during the micropropagation process. The amplified fragments of the micropropagated plantlets precisely matched those of the donor plants, confirming the maintenance of genetic uniformity in the micropropagation protocol. In conclusion, this study demonstrates that, successful micropropagation of Citrus lemon with retained genetic fidelity is of immense significance for large-scale multiplication and commercial production.
