Variety Pusa Basmati Research Articles

THE EFFECT OF COVID-19 ON THE VALUE CHAIN OF PADDY AND CARROT VARIETY OF IARI

The arrival of COVID-19 was no less than any apocalypse for the global economy. The growth of global GDP spiralled down to a very low point. The condition in India was no exception to it. Almost all the sectors of the economy were severely affected. Notwithstanding this, agriculture emerged as a bright spot in the dark horizon. Even though the growth rate was positive, there were serious impediments in the commodity value chain and agricultural marketing. Paucity in input supply, restriction in movement, closure of market, reduced demand of food products brought challenges that existed never before. In this backdrop, a study was conducted to find out the effects of COVID-19 on the value chain and marketing of Paddy Commercial variety Pusa Basmati 1121 and Carrot Commercial variety Pusa Rudhira. The research design was ex-post facto in nature and a total number of 160 farmers and 50 stakeholders including exporters, commission agents, wholesalers, and millers were consulted for the study. The locale of the study was Soodhna (U.P), Sangohi (Haryana), Khwajpur (U.P) and Nizampur (New Delhi). From extant review of literature, pilot study and discussion with experts, several constraints faced by the value chain stakeholders were identified and rank ordered using Rank Based Quotient Method (R.B.Q). Results showed that in case of farmers "difficulty in getting buyer, price fluctuation and wastage" were among the top constraints, and for stakeholders such as millers, wholesalers and retailers, "unavailability of labour and price fluctuations" were the causes of concern. Moreover, for exporters "hike in freight charges" was the most severe constraint. The findings of the study could highlight the vulnerable areas in the commodity value chain which ought to get due attention in policy making and better preparedness for future events of similar nature.

An Insight Into Pentatricopeptide-Mediated Chloroplast Necrosis via microRNA395a During Rhizoctonia solani Infection.

Sheath blight (ShB) disease, caused by Rhizoctonia solani, is one of the major biotic stress-oriented diseases that adversely affect the rice productivity worldwide. However, the regulatory mechanisms are not understood yet comprehensively. In the current study, we had investigated the potential roles of miRNAs in economically important indica rice variety Pusa Basmati-1 upon R. solani infection by carrying out in-depth, high-throughput small RNA sequencing with a total data size of 435 million paired-end raw reads from rice leaf RNA samples collected at different time points. Detailed data analysis revealed a total of 468 known mature miRNAs and 747 putative novel miRNAs across all the libraries. Target prediction and Gene Ontology functional analysis of these miRNAs were found to be unraveling various cellular, molecular, and biological functions by targeting various plant defense-related genes. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to validate the miRNAs and their putative target genes. Out of the selected miRNA-specific putative target genes, miR395a binding and its cleavage site on pentatricopeptide were determined by 5’ RACE-PCR. It might be possible that R. solani instigated chloroplast degradation by modulating the pentatricopeptide which led to increased susceptibility to fungal infection.

Loss of Function of OsFBX267 and OsGA20ox2 in Rice Promotes Early Maturing and Semi-Dwarfism in γ-Irradiated IWP and Genome-Edited Pusa Basmati-1.

Targeted mutagenesis is now becoming the most favored methodology to improve traits in popular rice cultivars selectively. Understanding the genetic basis of already available mutants could be the first step in designing such experiment. Improved White Ponni (IWP), a popularly grown South Indian rice variety, was subjected to γ irradiation to develop WP-22-2, an M6 line superior in semi-dwarfism, early flowering, and high yield, and it has grain qualities similar to those of IWP. The exogenous application of gibberellic acid (GA3) on WP-22-2 resulted in the elongation of shorter internodes to a level similar to IWP. The expression profiling of six genes regulating plant height showed their differential expression pattern at different time points post GA3 treatment. Furthermore, the sequencing of WP-22-2 and IWP genomes revealed several single nucleotide polymorphisms (SNPs) and large-scale deletions in WP-22-2. The conversion of functional codons to stop codons was observed in OsGA20ox2 and OsFBX267, which have been reported to have roles in regulating semi-dwarfism and early flowering, respectively. The loss of function of OsGA20ox2 and OsFBX267 in WP-22-2 resulted in reduced plant height as well as early flowering, and the same has been confirmed by editing OsGA20ox2 in the rice variety Pusa Basmati1 (PB1) using the CRISPR-Cas9 approach. The targeted editing of OsGA20ox2 in PB1 conferred shorter plant height to the edited lines compared with the wild type. Altogether, the study provides evidence on mutating OsGA20ox2 and OsFBX267 genes to develop early maturing and semi-dwarf varieties that can be released to farmers after functional characterization and field trials.

Identification and expression analysis of pathogenicity-related genes of Rhizoctonia solani anastomosis groups infecting rice.

The online version contains supplementary material available at 10.1007/s13205-021-02934-1.

Population buildup of yellow stem borer, Scirpophaga incertulas (Walker) of basmati rice in relation to abiotic factors

The population buildup of yellow stem borer, Scirpophaga incertulas was experimented during Kharif, 2018 and 2019 at thr CRC farm of Sardar Vallabhbhai Patel University of Agriculture and Technology, Meerut, U.P. The infestation of stem borer was recorded on variety Pusa Basmati-1 at weekly intervals from 32nd standard week and continued till the 43rd standard week and 33rd standard week and continued till the 43rd standard week during Kharif 2018 and 2019, respectively. The infestation of stem borer recorded as dead hearts /white ear heads and increased from third week of August and reached its peak (13.40%) in 41st standard week (Second week of October) and reached its peak (12.35%) in 36th standard week (second week of September) during Kharif 2018 and 2019, respectively.

Effect of different sources and application mode of zinc on growth, yield attributing characters and yield of rice variety Pusa basmati 1121 in sandy loam soil

The kharif season experiment comprised of twelve treatments in randomized block design with three replications consisting four levels of zinc sources (Zinc Sulphate, Mono Zinc suphate, Chelated Zinc and micronutrient mixture) with two mode of application (soil and foliar) with recommended NPK @120:60:60 and vermicompost @ 3t ha-1+ RDF thus, making 36 treatment combinations. The experimental soil was low in organic carbon and available nitrogen and medium in phosphorus and higher in potassium with slightly alkaline in pH. The kharif season experiment comprised of twelve treatments in randomized block design with three replications consisting four levels of zinc sources (Zinc Sulphate, Mono Zinc suphate, Chelated Zinc and micronutrient mixture) with two mode of application (soil and foliar) with recommended NPK @120:60:60 and vermicompost @ 3t ha-1+ RDF thus, making 36 treatment combinations. The experimental soil was low in organic carbon and available nitrogen and medium in phosphorus and higher in potassium with slightly alkaline in pH.

Effect of Boron on growth, yield and yield attributing characters of Rice Crop, (Oryza sativa L.)

A field investigation was carried out during Kharif seasons of 2012-2013 at Udai Pratap Autonomous College Varanasi (U.P). To the effect of six treatment of T0- Control, T1- B at 1 kg ha-1 + NPK at RD, T2- B at 1.5 kg ha-1 + NPK at RD, T3- B at 2 kg ha-1 + NPK at RD, T4- B at 2.5 kg ha-1 + NPK at RD and T5- B at 3 kg ha-1 + NPK at RD (Recommended dose) on rice crop (Oryza sativa L.) variety Pusa Basmati-1. The boron on growth and yield attributing characters rice crop significantly with the application of B at 2.5 kg ha-1 + NPK at RD (120 : 60 : 60 kg ha-1) The use of RD only but in application of boron improved the growth and yield attributing characters rice crop.

Mapping the Salt Stress-Induced Changes in the Root miRNome in Pokkali Rice.

A plant’s response to stress conditions is governed by intricately coordinated gene expression. The microRNAs (miRs) have emerged as relatively new players in the genetic network, regulating gene expression at the transcriptional and post-transcriptional level. In this study, we performed comprehensive profiling of miRs in roots of the naturally salt-tolerant Pokkali rice variety to understand their role in regulating plant physiology in the presence of salt. For comparisons, root miR profiles of the salt-sensitive rice variety Pusa Basmati were generated. It was seen that the expression levels of 65 miRs were similar for roots of Pokkali grown in the absence of salt (PKNR) and Pusa Basmati grown in the presence of salt (PBSR). The salt-induced dis-regulations in expression profiles of miRs showed controlled changes in the roots of Pokkali (PKSR) as compared to larger variations seen in the roots of Pusa Basmati. Target analysis of salt-deregulated miRs identified key transcription factors, ion-transporters, and signaling molecules that act to maintain cellular Ca2+ homeostasis and limit ROS production. These miR:mRNA nodes were mapped to the Quantitative trait loci (QTLs) to identify the correlated root traits for understanding their significance in plant physiology. The results obtained indicate that the adaptability of Pokkali to excess salt may be due to the genetic regulation of different cellular components by a variety of miRs.

Identification of microRNA-target modules from rice variety Pusa Basmati-1 under high temperature and salt stress.

High temperature and salinity stress are major factors limiting the growth and productivity of rice crop on a global scale. It is therefore an essential prerequisite to understand the molecular genetic regulation of plant responses to dual stresses. MicroRNAs (miRs) are recognized as key controllers of gene expression which act mainly at the post-transcriptional level to regulate various aspects of plant development. The present study attempts to investigate the miR circuits that are modulated in response to high temperature and salinity stress in rice. To gain insights into the pathway, preliminary miR profiles were generated using the next-generation sequencing (NGS) datasets. The identified molecules were filtered on the basis of fold differential regulation under high temperature, and time kinetics of their expression under the two individual stresses was followed to capture the regulatory windows. The analysis revealed the involvement of common miR regulatory nodes in response to two different abiotic stresses, thereby broadening our perspective about the stress-mediated regulatory mechanisms operative in rice.

Microprojectile based particle bombardment in development of transgenic indica rice involving AmSOD gene to impart tolerance to salinity

Genetic transformation of Pusa Basmati 1, a high yielding scented indica rice variety has been successfully achieved using AmSOD gene housed in a binary vector pSFSOD1 under the transcription control of ubiquitin promoter and nos terminator through microprojectile bombardment for increased tolerance towards salinity stress. Initially the protocol was optimized with binary vector pCAMBIA1305.2 harbouring GUS Plus™ reporter gene involving embryogenic calli developed from mature embryos of three rice varieties viz., Pusa Basmati 1, Taraori Basmati and one new plant type-super rice variety- IR70485–15–3-2 using PDS He 2000. The reporter gene showed significant GUS expression, which indicates maximum transformation efficiency at transient level under optimized parameters such as helium pressure-1100 psi, internal vacuum pressure-28″, flight distance-9 cm, microcarrier-1 μM diameter, particle volume-15 μl and microcarrier launch assembly at 6 cm for efficient bombardment of the embryogenic calli. The bombarded calli were transferred to MS medium supplemented with mannitol (0.2 M) and sorbitol (0.2 M) for 4 h, found to be most appropriate osmoticum in bombardment shock recovery and their proliferation. This best optimized protocol was used to dispense AmSOD gene for development of transgenic lines of the most in vitro culture responsive variety Pusa Basmati 1 to impart tolerance to salinity. Putative transgenic plants were developed from the bombarded calli, which were acclimatized, hardened and grown to maturity and analyzed by using molecular, physiological and biochemical assays to determine the introgression of the transgene into the variety. Further, inheritance study showed typical Mendelian ratio for monogenic dominant transgene in the segregating T2 generation. Among progenies tested, PBT 3–2, PBT 6–4 and PBT 8–9 were found to be appreciably tolerant to salinity stress with high yield at different growth stages, which prospects amply for more production and profitability to attain farm prosperity by large-scale cultivation of transgenic Pusa Basmati 1 on saline soils.

Introgression of sheath blight disease tolerance from the transgenic rice event Pusa Basmati1-CG27 to the variety White Ponni through backcross breeding

Sheath blight disease caused by Rhizoctonia solani is a major constraint for rice production. In order to obtain transgenic resistance against sheath blight disease, an indica rice variety Pusa Basmati1 (PB1) was transformed with constitutively express chitinase (chi11) and β-1,3-glucanase (GLU) genes. A single-copy transgenic PB1-CG27 showed a high level of tolerance to R. solani. Here, we report the introgression of sheath blight tolerance from PB1-CG27 into a non-basmati rice variety, White Ponni (WP) through backcross breeding. In each of the four backcross generations, the presence of the transgene was checked by hygromycin phosphotrans-ferase (hph) and βglucuronidase (gus) gene-specific foreground selection markers present in the T-DNA of the PB1-CG27 event. RAPD marker-based background selection was done to choose a BC4F1 plant that was similar to the WP recurrent parent. Morphological characters of the introgressed BC4F4 plants matched those of the recurrent WP parent. RT-PCR and western blot analyses confirmed the constitutive expression of the chi11 and GLU genes in the introgressed homozygous BC4F3 plants. Bioassay with R. solani a high level disease tolerance (43% disease reduction) revealed over the control WP plants.

SINGLE AND COMBINED EFFECTS OF THREE FUSARIUM SPECIES ASSOCIATED WITH RICE SEEDS ON THE SEVERITY OF BAKANAE DISEASE OF RICE

Fusarium spp. associated with susceptible rice variety Pusa Basmati 1121 were isolated. Based on translation elongation factor 1α (TEF-1α) Fusarium spp. were identified as F. fujikuroi, F. proliferatum, F. verticillioides (F. moniliforme), F. sacchari, F. pseudonygamai, F. thapsinum and F. andiyazi. Pathogenicity test conducted on susceptible variety Pusa Basmati 1121 confirmed that only F. fujikuroi, F. proliferatum and F. verticillioides produced bakanae disease as elongation, rotting or both types of symptoms. Gibberellic acid was exclusively extracted from the isolates of F. fujikuroi and it ranged from 450 μg/g to 4361 μg/g of mycelium. Gibberellic acid content was positively correlated (r=0.79) with elongation symptom of the disease. Fusaric acid was present in all the species of Fusarium, ranging from 60 μg/g to 2920 μg/g of mycelium. However, significant correlation between fusaric acid content and disease severity could not be established. Co-inoculations of pathogens showed significant reduction in percent seed germination and increased disease severity compared to pathogen inoculated individually. Therefore, present investigation will be helpful to understand the effect of different Fusarium spp. in bakanae disease etiology.

Effect of integrated nutrient management on yield and phosphorus availability of aromatic rice (Oryza sativa L.) in inceptisol of eastern U. P.

Field experiment for quality rice production of scented rice variety Pusa Basmati and NDR-Lalmati was conducted on Students Instructional Farm, Narendra Deva university of Agriculture and Technology, Narendra Nagar, Faizabad during Kharif season of 2013 and 2014. The treatment were included inorganic and organic combinations viz., T 1 : RDF NPK (100:50:50) × V 1 , T 2 : RDF NPK + 5 tons FYM/ha × V 1 , T 3 : 75 per cent RDF NPK+25 per cent N with FYM × V 1 , T 4 : 75 per cent RDF NPK+25 per cent Nwith green manure × V 1 , T 5 : 50 per cent RDF +25 per cent FYM-N+25 per cent GM-N × V 1 , T 6 : 20 tons FYM/ha × V 1 , T 7 : RDF NPK (100:50:50) × V 2 , T 8 : RDF NPK + 5 tons FYM/ha × V 2 , T 9 : 75 per cent RDF NPK+25 per cent N with FYM × V 2 , T 10 : 75 per cent RDF NPK+25 per cent N with green manure × V 2 , T 11 : 50 per cent RDF +25 per cent FYM-N+25 per cent GM-N × V 2 , T 12 : 20 tons FYM/ha × V 2 . The treatments were replicated thrice in Factorial Randomized Block Design. The variety Pusa Basmati recorded higher grain and straw yield as compared to NDR-Lalmati in both year of investigation. Maximum grain and straw yield of aromatic rice was recorded under integrated nutrient management of 100 per cent NPK + 5 t FYM followed by treatment T 1 and T 4 in both the years of investigation.

Evaluation of virus resistance and agronomic performance of rice cultivar ASD 16 after transfer of transgene against Rice tungro bacilliform virus by backcross breeding.

Severe losses of rice yield in south and southeast Asia are caused by Rice tungro disease (RTD) induced by mixed infection of Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV). In order to develop transgene-based resistance against RTBV, one of its genes, ORF IV, was used to generate transgenic resistance based on RNA-interference in the easily transformed rice variety Pusa Basmati-1, and the transgene was subsequently introgressed to rice variety ASD 16, a variety popular in southern India, using transgene marker-assisted selection. Here, we report the evaluation of BC3F4 and BC3F5 generation rice plants for resistance to RTBV as well as for agronomic traits under glasshouse conditions. The BC3F4 and BC3F5 generation rice plants tested showed variable levels of resistance, which was manifested by an average of twofold amelioration in height reduction, 1.5-fold decrease in the reduction in chlorophyll content, and 100- to 10,000-fold reduction in the titers of RTBV, but no reduction of RTSV titers, in three backcrossed lines when compared with the ASD 16 parent. Agronomic traits of some of the backcrossed lines recorded substantial improvements when compared with the ASD 16 parental line after inoculation by RTBV and RTSV. This work represents an important step in transferring RTD resistance to a susceptible popular rice variety, hence enhancing its yield in areas threatened by the disease.

Ectopic expression of PgRab7 in rice plants (Oryza sativa L.) results in differential tolerance at the vegetative and seed setting stage during salinity and drought stress.

In this work, we have overexpressed a vesicle trafficking protein, Rab7, from a stress-tolerant plant, Pennisetum glaucum, in a high-yielding but stress-sensitive rice variety Pusa Basmati-1 (PB-1). The transgenic rice plants were tested for tolerance against salinity and drought stress. The transgenic plants showed considerable tolerance at the vegetative stage against both salinity (200mM NaCl) and drought stress (up to 12days after withdrawing water). The protection against salt and drought stress may be by regulating Na+ ion homeostasis, as the transgenic plants showed altered expression of multiple transporter genes, including OsNHX1, OsNHX2, OsSOS1, OsVHA, and OsGLRs. In addition, decreased generation and maintenance of lesser reactive oxygen species (ROS), with maintenance of chloroplast grana and photosynthetic machinery was observed. When evaluated for reproductive growth, 89-96% of seed setting was maintained in transgenic plants during drought stress; however, under salt stress, a 33-53% decrease in seed setting was observed. These results indicate that PgRab7 overexpression in rice confers differential tolerance at the seed setting stage during salinity and drought stress and could be a favored target for raising drought-tolerant crops.

Productivity and profitability of aerobic rice (Oryza sativa) as influenced by varieties and integrated nitrogen management

A field experiment was conducted during the kharif seasons of 2009 and 2010 at Indian Agricultural Research Institute, New Delhi to study productivity and profitability of aerobic rice as influenced by varieties and integrated nitrogen management. The experimental treatments included 2 rice varieties, viz. Pusa Basmati 1 and Pusa Basmati 1121 and 8 integrated nitrogen management practices, viz. N control (N0); 100% RDN (120 kg N/ha through urea); 75% RDN + 25% N through farmyard manure; 75% RDN + 25% N through green manuring; 75% RDN + 25% N through biofertilizers; 75% RDN + 25% N through vermicompost; 100% N through FYM+GM+BF+VC and 100% N through FYM+GM+BF+VC+ZnSO4. The results showed that rice variety Pusa Basmati 1 out performed Pusa Basmati 1121 in yield attributes, grain yield and harvest indices. Application of recommended dose of nitrogen (RDN) through integrated nitrogen management (INM) approach significantly increased the crop growth parameters, yield attributes, grain as well straw yields and net returns over other INM approaches. Treatment with 100% N by FYM+GM+BF+VC+Zn showed highest crop growth and yield attributes, grain and straw yield and net returns as compared to other integrated nitrogen management treatments, but this treatment was closely followed by 100% N by FYM+GM+BF+VC and 75% RDN + 25% VC during both the years.

Occurrence, identification and pathogenicity of Fusarium species associated with bakanae disease of basmati rice in India

The incidence of bakanae disease of rice was studied in the basmati growing states of Punjab, Haryana, Uttar Pradesh and Uttarakhand in India. Maximum disease incidence (20 %) was observed from the Karnal district of Haryana and Bulandshar, Ghaziabad and Mathura districts of Uttar Pradesh in rice variety Pusa Basmati 1121 and Pusa Basmati 1509. Disease was also observed in other popular basmati rice varieties of the country including Pusa Basmati 6, Pusa 2511, CSR 30, Pakistani Basmati and Sarbati with the incidence of 0.5 % to 10 %. One hundred and twenty-six isolates of Fusarium spp. were isolated from symptomatic plants and were characterized morphologically and 42 isolates were characterized at the molecular level. Out of 42 isolates characterized based on translation elongation factor 1 α (TEF- 1α), 41 were identified as Fusarium fujikuroi. Virulence analysis performed on rice variety Pusa Basmati 1509 categorized the isolates as moderately virulent (37.3 %), virulent (34.1 %) and highly virulent (28.6 %). Results indicated that F. fujikuroi is predominant in symptomatic diseased plants and a management programme should be developed by considering the prevalence and virulence of the pathogen in the area of intended cultivation.

Integration of Seed Treatments, Seedling Dip Treatments and Soil Amendments for the Management of Bakanae Disease in Paddy Variety Pusa Basmati 1121

Integration of Seed Treatments, Seedling Dip Treatments and Soil Amendments for the Management of Bakanae Disease in Paddy Variety Pusa Basmati 1121

Development and evaluation of near-isogenic lines for major blast resistance gene(s) in Basmati rice.

A set of NILs carrying major blast resistance genes in a Basmati rice variety has been developed. Also, the efficacy of pyramids over monogenic NILs against rice blast pathogen Magnaporthe oryzae has been demonstrated. Productivity and quality of Basmati rice is severely affected by rice blast disease. Major genes and QTLs conferring resistance to blast have been reported only in non-Basmati rice germplasm. Here, we report incorporation of seven blast resistance genes from the donor lines DHMASQ164-2a (Pi54, Pi1, Pita), IRBLz5-CA (Pi2), IRBLb-B (Pib), IRBL5-M (Pi5) and IRBL9-W (Pi9) into the genetic background of an elite Basmati rice variety Pusa Basmati 1 (PB1). A total of 36 near-isogenic lines (NILs) comprising of 14 monogenic, 16 two-gene pyramids and six three-gene pyramids were developed through marker-assisted backcross breeding (MABB). Foreground, recombinant and background selection was used to identify the plants with target gene(s), minimize the linkage drag and increase the recurrent parent genome (RPG) recovery (93.5-98.6%), respectively, in the NILs. Comparative analysis performed using 50,051 SNPs and 500 SSR markers revealed that the SNPs provided better insight into the RPG recovery. Most of the monogenic NILs showed comparable performance in yield and quality, concomitantly, Pusa1637-18-7-6-20 (Pi9), was significantly superior in yield and stable across four different environments as compared to recurrent parent (RP) PB1. Further, among the pyramids, Pusa1930-12-6 (Pi2+Pi5) showed significantly higher yield and Pusa1633-7-8-53-6-8 (Pi54+Pi1+Pita) was superior in cooking quality as compared to RP PB1. The NILs carrying gene Pi9 were found to be the most effective against the concoction of virulent races predominant in the hotspot locations for blast disease. Conversely, when analyzed under artificial inoculation, three-gene pyramids expressed enhanced resistance as compared to the two-gene and monogenic NILs.

Exogenous Application of 28-Homobrassinolide Modulates the Dynamics of Salt and Pesticides Induced Stress Responses in an Elite Rice Variety Pusa Basmati-1

Brassinosteroids play an essential role in regulating various aspects of plant growth and development as well as adaptation to various environmental stresses. The present work provides an analysis of the response of various stress markers upon exogenous application of 28-homobrassinolide (HBL) on Pusa Basmati-1, a commercially important rice variety, under salt and pesticide (Chlorpyrifos and Imidacloprid) stress. Rice seeds treated with HBL were analyzed for various growth parameters, protein, proline and malondialdehyde content (MDA), antioxidant enzyme activities, and their gene expression analysis (Cu/Zn-SOD, Fe-SOD, Mn-SOD, APX, CAT, and GR) in the presence or absence of salt and pesticide stress. Stress-induced reduction in growth, protein, and chlorophyll content and enhancement of proline and MDA content of seedlings was observed. The exogenous application of HBL resulted in the improvement of growth parameters as well as protein and proline content. MDA content decreased significantly under the effect of HBL treatment both under stress and control conditions. HBL treatment also enhanced the activity of various antioxidant enzymes which corroborated with the reduced accumulation of O 2 ⋅- and H2O2 under the effect of salt and pesticides. The differential response of various isoforms of SOD under the effect of HBL and stress treatments was observed under salt and among different pesticide treatments. From this study, the potent activity of HBL in stress mitigation in response to salt and pesticide treatment in rice is established.