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Open Access
GENE EXPRESSION STUDIES FOR LOW TEMPERATURE TOLERANCE AT THE BOOTING STAGE IN RICE
(2023) Bora, Smriti Shyamolee; Baruah, Akhil Ranjan
Rice (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- 12.5C 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.
Open Access
STUDY ON GENETIC FIDELITY IN TISSUE CULTURED RAISED PLANTLETS OF CITRUS LIMON (ASSAM LEMON)
(2023) Das, Ratnakishore; Sen, Priyabrata
Citrus 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.
Open Access
MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION OF VESICULAR ARBUSCULAR MYCORRHIZA ASSOCIATED WITH LEGUMES
(2023) Kudve, Rajkumar Laxman; Nath, Tankeswar
Plant health and soil fertility are largely influenced by nos. of associations between plant and microbes. Among these, the association between Vesicular Arbuscular Mycorrhiza (VAM) and the plant is well-known. VAM fungi create symbiotic association with the roots of plants and increase plant growth largely through phosphorus nutrition. The International Collection of Vesicular Arbuscular Mycorrhiza (INVAM), University of Kansas, U.S. reported that around 160 species of Arbuscular Mycorrhizal Fungi (AMF) have been described by spore morphology. However, in nature there may be great variation of spore‟s morphology within a VAM species. Hence, it is necessary to conduct research on VAM to analyze genus diversity in the soil and for their use as biofertilizer. However, VAM have not been studied extensively at morphological and molecular level by regional diversity like other beneficial microorganisms. Again, the molecular level identification is necessary due to most of the mycorrhizal species are structurally similar, and hence they cannot be differentiated on the basis of their morphological observations. Therefore, in this present study an attempt had been made to isolate and characterize VAM indigenous to Assam, at morphological and molecular levels. For this, VAM spore had been isolated from the rhizospheric soil of two legume crops, Dhaincha (Sesbania bispinosa) and Subabul (Leucaena leucocephala) from ICR farm AAU, Jorhat. At first, morphological identification of the isolated VAM fungi was done under microscope. Spore, sporocarps and hyphal structure were observed by mounting with polyvinyl alcohol-lactic acid-glycerol (PVLG). On the basis of morphological observations (spore color, spore shape and hyphal color) they are supposed to come under the genus, Glomus, Gigaspora, Acaulospora and Scutellospora. For molecular characterization, the isolated VAM fungal spores inoculated with maize as the trap plant in pot culture technique. Further, the presence of arbuscule, vesicle and hyphae of VAM in infected roots of maize seedling were studied under microscope after staining with trypan blue. The study was further extended to molecular characterization by using both universal fungal primers [NS1(F) and NS2(R)] as well as mychorrhyza specific primers [AML1 (F) and AML2 (R)]. Finally, the mycorrhizal isolates were identified as Glomus, Gigaspora, Acaulospora and Scutelospora species. Thus, the tentative classification of AMF done on the basis of morphological characteristics was supported by the molecular characterizations. This was a preliminary study of rhizospheric AM fungi associated with legumes. However, this study can further be extended to study the diversity of plant associated mycorrhizal fungi in Assam soils which will help to develop mycorrhiza based biofertilizer specific to this region. Furthermore, it will give a picture of mycorrhizal diversity of Assam, understanding of which is important to maintain productivity and stability of ecosystem of this region.
Open Access
Molecular basis of non -host resistance to Helicoverpa armigera
(2023) Kanth, Deepak Ranjan; Acharjee, Sumita
The complex molecular mechanism underlying resistance in non-host crops against Helicoverpa armigera is yet to be elucidated. Non-host resistance exhibits remarkable efficacy and broad-spectrum capabilities in countering potential pests, hence, has proven to be a valuable resource for understanding the basis of resistance. Our lab previously studied the molecular mechanism of legume (chickpea and pigeon pea) interactions with H. armigera using transcriptomic and proteomics approaches. Several cell receptor-like kinases (RLKs)/receptor-like proteins (RLPs) and defense-related genes were identified in these studies. In this present investigation, an effort has been made to understand the changes in expression patterns of a few selected cell receptor-like kinases (RLKs)/receptor-like proteins (RLPs), and defense-related genes in response to simulated herbivory (Oral Secretions/OS) and exogenous application of hormones such as Jasmonic acid (JA), Salicylic acid (SA) and Ethephon (ET). The generation of Reactive Oxygen Species (ROS) in samples after 12 h of oral secretion (OS) treatment confirmed that various elicitors were present in OS for effective induction of defense in groundnut. Gene expressing analysis showed significant (>5-fold) upregulation of the Proline-rich RLK PERK 15 gene due to the application of OS. Moreover, the expression of defense-related genes such as Endoglucanase 8-like, PR10, ABA-responsive protein ABR17, Galactose-binding lectin, and Cytochrome P450 90B1 also showed upregulation after 24 h of OS treatment. However, the expression of the above genes was downregulated after 48 h of treatment. It was also observed that the expression of lysM domain RLK 3, ABA-responsive ABR17, and PR10 genes was regulated by JA and ET pathways while galactose-binding lectin gene expression was mediated by the SA pathway. The expression of the endoglucanase 8-like gene showed an antagonistic regulation by JA and SA. Moreover, the trypsin inhibition (TI) assay revealed the expression of TI in the treated samples. Thus, the above results suggest that during the interaction H. armigera, elicitors present in OS are perceived by lysM domain RLK3 receptor in a non-host, groundnut, which activated the downstream signaling pathways to upregulate the expression of various defense-related genes. It would be interesting to study the role of galactose-binding lectin in the non-host defense against H. armigera. The present study shed light on the non-host resistance mechanism using the groundnut-Helicoverpa interactions as a model, which revealed changes in the groundnut gene expression pattern due to simulated herbivory.
Open Access
Expression analysis of pathogenesis-related (PR) genes in Brassica rapa upon Alternaria brassicicola infection
(2023) R, Aryasree; Bhorali, Priyadarshini
Alternaria blight caused by Alternaria brassicicola is the most widespread fungal disease of oilseed Brassicas. Conventional breeding to develop Alternaria resistant cultivars has not been successful due to non-availability of suitable resistance sources. For the development of resistant cultivars, identification of important defense or resistance-related genes is needed. Plants activate different biological pathways upon infection by pathogens and a large number of defense-related genes are modulated during such interactions locally as well as systemically. In a previous study, RNAseq of a wild relative of Brassica, Sinapis alba, was carried out in which several defense related genes were found to be highly upregulated during Alternaria infection. Based on the results of this study, the present research was undertaken to investigate and validate the gene expression patterns of selected gene candidates in the susceptible cultivar B. rapa var. Toria (TS-38) in order to check the function of these genes in conferring resistance. To initiate the study, infected tissues of a highly susceptible Toria variety TS-38, showing characteristic symptoms of Alternaria blight were collected for isolation and purification of the pathogen. On the basis of conidial morphology as observed through microscopic studies, the pathogen was identified to be Alternaria brassicicola. Molecular detection of the fungus was carried out by amplification using ITS primers followed by sequencing. Pathogenicity test was done through detached leaf assay and seedling inoculation test and confirmation was done by testing the Koch postulates. After pathogen inoculation, ROS generation and proline accumulation were detected during the biotic stress. Staining with trypan blue helped to visualise necrosis in response to A. brassicicola infection in the leaf tissues. Finally, total RNA was isolated from the B. rapa inoculated (and control) leaf tissues followed by cDNA preparation for real time qRT-PCR analysis of gene expression. Primers available from the previous study, which were designed using S. alba sequences of some important gene candidates, including PR and resistance-related genes were used for the expression analysis. The results depict that all the PR genes were upregulated at lower levels at 24 hpi. Later, although the expression increased towards 48 hpi, it gradually decreased. The resistance related genes also showed upregulation with peak expression at 48 hpi, but later downregulated. Interestingly, one disease resistance (DR) gene showed downregulation across all the time points in the susceptible cultivar and and could be an important gene responsible for resistance. Thus, all the genes are clearly implicated to have a role in active defense responses. As a future prospect, complete functional characterization of such genes could be done as they would serve as important potential candidates for developing disease resistant varieties of Brassica.
Open Access
Molecular cloning and in silico characterization of linalool synthase gene from Cymbopogon winterianus
(AAU, Jorhat, 2018-07) Saha, Oliva; Sen, Priyabrata
Citronella (Cymbopogon winterianus), an aromatic and medicinal grass from Poaceae family, is grown for the commercial and industrial purpose. It has the large repository of isoprenoid compounds known for its anti-tumoral, anti-bacterial, anti-fungal, anti-viral, anticancerous, detoxifying and natural insect repellent properties. Out of the different components of citronella oil, linalool (3,7-Dimethylocta-1,6-dien-3-ol) is one of the significant aromatic ingredient. Formation of linalool is catalysed by linalool synthase (LIS) which is encoded by linalool synthase gene. This gene has been widely studied in many medicinal plants along with sorghum, rice, maize, setaria, etc.However, till date, no studies on molecular cloning, characterization and tissue-specific expression profiling of LIS gene from C. winterianus (CwLIS) has been reported.Therefore, the present study aims at cloning and characterization of full-length cDNA of LIS gene from C. winterianus. The full-length sequence of CwLIS was obtained by primer walking using several pairs of degenerate primers and the sequence fragments were aligned to acquire full length sequence. Cloning of the desired gene (1758bp) was performed using TOPO TA vector (3.9kb), and the transformation was done in E. coli DH5α competent cells. The sequence analysis revealed 1758 bps cDNA, which has a Coding Sequence (CDS) of 1422 bps that encodes a protein of 473 amino acids. The domain analysis revealed that CwLIS is a single domain protein under terpene synthase superfamily. The multiple sequence alignment (MSA) based on PSI-BLAST search against RefSeq with 62% sequence identity revealed the presence of two aspartate-rich regions, which are supposed to coordinate 3 Mg2+ ions. The phylogenetic analysis revealed that the CwLIS is closely related to Sorghum bicolor linalool synthase. To understand the molecular mechanism behind Mg2+ and linalool binding, first, the 3D model of CwLIS was generated and validated with their stereo chemical parameters. Further, to find out the expression profile of CwLIS in different tissues, Reverse Transcriptase-PCR was performed and the results revealed a higher expression in leaf sheath followed by leaf, root and flower and further conformed by qRTPCR analysis. The result from the present study provide basic information for further research about linalool synthase and comprehensive sequence resource for study, such as gene expression, genomics and functional genomics in Cymbopogon winterianus.
Open Access
Assessment of Genetic Diversity For Ideal Plant Architecture in Rice Cultivars of Assam With Special Importance to Aromatic Group
(AAU, Jorhat, 2018-08) Debnath, Neelakshi; Baruah, A.R.
The development of super rice to break the yield plateau is one of the goals for rice breeding in recent years. Ideal plant architecture (IPA), which is under polygenic control, has been proposed as means to enhance rice yield potential. A study directed towards assessing the presence of desired alleles for IPA related genes/quantitative trait loci (QTL) in rice cultivars grown in diverse agroecological situations, specifically the aromatic rice of Assam (joha rice) that usually bears weaker plant type should be of prime importance. The present study was conducted to detect allelic variation for IPA in traditional, high yielding and joha rice cultivars of Assam and to compare nucleotide sequence variation for aroma gene in cultivars possessing IPA. A total of 80 cultivars comprising sali, ahu, boro and joha rice of Assam were assessed for markers linked to five IPA related QTLs (Sol1, PAY1, IPA1, Gn1 and LAZY1). Although a total of 15 markers (M3, M6, M8, M10 for Sol1; RM339, RM223, SP5, SP7 for PAY1; RM149, RM1345, M4, M5 for IPA1; PD56, M265 for LAZY1; 16BPDEL for Gn1) were employed to check for polymorphism, only five markers (GN1, M10, M265, RM 1345, SP5) showed reproducible and polymorphic results. The Gn1 and Sol1 were most abundant in cultivars as evident from the observation that 46 numbers of cultivars harboured desired alleles for Gn1 and Sol1 followed by PAY1( 31 cultivars), LAZY1 (12 cultivars) and IPA1(12 cultivars). Among HYVs, the varieties such as Ranjit, Mahsuri and Bahadur were found to possess four numbers of desired alleles, however, IPA1 allele was other than the expected. Among Joha cultivars, Bengoli joha, Goalporia joha, Kartica joha were with the maximum of four numbers of positive alleles. As Ranjit missed the expected size of the IPA1 allele, differential expression for the gene in Ranjit and one cultivar possessing the desired IPA1 allele (Chakaw Poireton) was conducted. It revealed that although Ranjit did not possess the desired IPA1, however, the expression pattern revealed that 25-30 fold more accumulation of transcripts for the gene in Ranjit than Chakaw Poireton, suggesting the functional differences in the coding regions. The allele specific analysis for aroma gene, badh2 revealed that the markers could efficiently group 53.66% of the cultivars corresponding to the existing phenotype, indicating the power of the marker to be utilized in a large germplasm screening for aroma. The sequencing for badh2.hapG9 detected a non-synonymous substitution (AG) which was not being reported elsewhere; however, novelty of which needed to be confirmed and validated. The study will enhance our knowledge to formulate breeding strategies for combing IPA with aroma.
Open Access
Optimization of an in vitro regeneration and transformation protocol in Khasi Mandarin
(AAU, Jorhat, 2017-07) Das, Panchashree; Sen, Priyabrata
Citrus species are the most widely grown fruit crops within the whole world. India is the fourth largest producer of orange in the world. North-Eastern India is considered as one of the centres of origin of many citrus species. Among them Khasi Mandarin is the most widely grown citrus species. According to Ministry of Agriculture and Irrigation, Govt. Of India, the yield of Khasi Mandarin is declining day by day drastically due to biotic and abiotic stress. Conventional breeding for overcoming this problem is limiting due to non-availability of resistant sources. Recent advances in genetic engineering have made it possible to incorporate desirable genes from alien sources to elite genotype mainly through Agrobacterium-mediated genetic transformation. Citrus cultivars vary in their response to in vitro organogenesis and genetic transformation. This results in need for a cultivarspecific optimization of an in vitro regeneration and transformation protocol. Most of the plant regeneration processes in citrus, through tissue culture, involve use of Cotyledon, epicotyl segment, shoot-tip, internode, root meristem as explants. A study was conducted to develop a regeneration and Agrobactetrium mediated transformation protocol for Khasi Mandarin using zygotic seedling as explants obtained from six-week-old in vitro grown seedlings. Modified MS media containing 1 mg/l BAP, 0.5mg/l NAA and 0.4mg/l Kinetin shows the best result for multiple shoot induction with an efficiency of 68%. The number of multiple shoots developed was on an average 5. The modified MS medium containing containing 0.25 mg/l BAP,0.5 mg/l NAA, 0.5 mg/l IBA shows best result for rooting with an efficiency of 82% with an average root length 2 cm. Zygotic explants with injured shoot tip were used as explant for transformation with Agrobacterium strain AGL1, harbouring plasmid pCAMBIA1301 containing hpt as selectable marker gene and gus as a reporter gene. Modified MS media containing 100mM Acetosyringone was fund to be most effective medium for co-cultivation. Regeneration and selection media containing 1 mg/l BAP, 0.5mg/l NAA, 0.4mg/l Kinetin and 30mg/l hygromycin and 250mg/l timentin shows the best result. In vitro regenerated shoots that survived upto 3rd selection cycle were considered as putative transformants. Some of the putative transformed shoots showed positive result for gus in PCR analysis. The present investigation is a preliminary study on optimization of an in vitro regeneration and transformation protocol in Khasi Mandarin. More and more concentrated effort is needed to establish a most efficient regeneration and transformation protocol considering various factors affecting genetic transformation and regeneration efficiency.
Open Access
HOST RANGE, INCIDENCE AND GENETIC VARIABILITY OF watermelon mosaic virus IN CENTRAL SPAIN
(AAU, Jorhat, 2018-01) Paswan, Ricky Raj; Acharjee, Sumita
Watermelon mosaic virus (WMV, Potyvirus) is an economically important pathogen common in cucurbits of temperate and Mediterranean regions worldwide. The presence of WMV in cucurbits in the Mediterranean basin has been known for decades. More recently, an emergent strain that causes more severe symptoms compared to classic strains has been recognized in France. The cultivation of melon is threatened by the spread of emergent strains of WMV. Diagnostic methods for detecting the host range, incidence and evolution of these emergent types are critical for developing control strategies to optimize agricultural production. In this study, next generation sequencing and RT-PCR approaches are combined to investigate the epidemiology of WMV in an agro-ecosystem of Central Spain. Four vegetation types, or habitats, including cultivated and adjacent land-use types were surveyed in the summer and autumn of 2015. Forty-three plant species were screened for WMV, 15 of which were WMV-positive across two habitats other than crops. The results indicated an increase in the extent of the WMVs known host range. The incidence of WMV ranged from 64% in Cucumis melo to 5% in a weed species, Datura Stramonium. Genetic analyses of the coat protein gene of 30 isolates from melon and 3 other ‘weed’ species sampled in crops showed population variation in nucleotide diversity, but pairwise fixation indices indicated negligible distinctions between them. Phylogenetic inferences showed both negligible and large branch length differences between isolates from different host species. When sequences of a number of different strains were added to the isolates from the melon crops, one clad clustered with an emergent group previously identified from elsewhere in Europe and Asia. This study reports the first instance of an emerging (EM) strain in Central Spain.

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