Annual Report 2019 ICAR-National Institute for Plant Biotechnology Lal Bahadur Shastri Centre, Pusa Campus New Delhi - 110012 Annual Report 2019 ICAR-National Institute for Plant Biotechnology Lal Bahadur Shastri Centre, Pusa Campus New Delhi - 110012 Published by Dr. Sarvjeet Kaur Director (In charge) ICAR-National Institute for Plant Biotechnology Pusa Campus, New Delhi - 110012 Tel.: 25848783, 25841787, 25842789 Fax: 25843984 Website: http://www.nipb.icar.gov.in Compiled and Edited by Dr. Sarvjeet Kaur Dr. Monika Dalal Dr. Prasanta Dash Dr. Rohini Sreevathsa Dr. Amitha C.R. Mithra S.V. Dr. Navin C. Gupta Dr. Deepak S. Bisht Dr. Nimmy M. S. Dr. Rampal S. Niranjan Correct Citation Annual Report 2019 ICAR-National Institute for Plant Biotechnology Pusa Campus, New Delhi - 110012 ii Preface ICAR-NIPB is a premier research institute of ICAR in the field of plant biotechnology with a mandate of undertaking basic plant molecular biology research for understanding molecular mechanisms underlying basic biology processes, development of capabilities of devising tools and techniques of biotechnology and genetic engineering for crop improvement, as also application of the knowledge of genomics for advancing agriculture development. Another significant mandate of the institute is to serve as a national lead center for plant molecular biology and biotechnology research and to create trained manpower in the area of plant biotechnology. The five mandated crops of NIPB are: rice, wheat, mustard, pigeon pea and chick pea. Research on addressing various aspects of crop improvement in these crops such as biotic-abiotic, stress tolerance and quality traits has been carried out in the period under report. The core research activities of our institute include, structural and functional genomics, mapping of QTLs and development of markers, transgenic development, collection/generation and maintenence of germplasm and mutant resources and bioprospecting of genes. The major achievements of the institute for the year 2019 include identification of 1 major QTL for seedling stage and 4 QTLs for reproductive stage salt tolerance in rice. The QTLs for anaerobic germination were identified in a mapping population from IR64/ NKSWR397 as well as though Genome Wide Association Studies in deep-water rice population of Assam. From wild rice collection, novel sources of submergence tolerance and resistance to brown spot pathogen were identified. From rice mutant resource, three stay-green mutants were identified and one of them showed better harvest index than Nagina 22 under drought stress. Based on large scale phenotyping of mutants at hotspots and multi-location testing, three mutants with high level of resistance against leaf and panicle blast were identified. Small RNA, lncRNAs and transcriptome analysis was carried out in rice for different traits such as deep water tolerance, nitrogen use efficiency, panicle blast resistance, sheath blight infection. Transgenic rice overexpressing Os06g10210 and Os04g0690500 were evaluated for abiotic stress tolerance and were found to be tolerant to drought, cold and salinity stress. To improve biological nitrogen fixation, transgenic rice that are able to support the formation of symbiosome compartments in rice roots for harbouring/ sequestering rhizobia and protecting them from plant defence system have been developed. Genome editing constructs for resistance to blast and fungal pathogen Rhizoctonia solani have been developed. For development of wheat varieties resistant to Bipolaris leaf spot, marker assisted backcross breeding is being carried out and QTLs for yellow rust resistance are also being introgressed in the promising leaf spot resistant lines. Three hundred diverse wheat genotypes were phenotyped for component traits of nitrogen use efficiency and genotyped by 35k SNP Breeder’s array. Two orthologs of AtNRT1.5, TaNRT2.1/2.2 and TaNAR2.2/2.2 were cloned and were shown to interact with each other using split-ubiquitin assay. Gene TaNHX1 from salt tolerant genotype Kharchia was validated in wheat. Transient expression analysis of TaPM19 Promoter deletion fragments was carried out in tobacco. Promising stress responsive genes, ZnClpB1 from Z. nummularia and EcDREB2A from finger millet were functionally iii validated in Nicotiana tabacum. RNA-seq analysis of hexaploid bread wheat and its diploid progenitors during grain development was carried out. ICAR-NIPB participated as a collaborator in the development of a drought-tolerant variety Pusa Chickpea 10216 (BGM 10216) released by IARI. Genome-wide association mapping in a panel of 402 chickpea genotypes for seed protein content led to identification of 23 gene- based SNPs, which exhibited significant phenotypic variance in seed protein content. Based on genome wide analysis of chick pea, seven true homologs of Arabidopsis SOS genes in chickpea were identified. The work on Fusarium wilt in chickpea has been initiated this year in the institute. The full-length mitochondrial genome of the chickpea Fusarium wilt pathogen, Fusarium oxysporum f. sp. ciceris (Foc) was assembled from publicly available whole-genome short reads Illumina data for further analysis of racial diversity of Foc in different agroecological zones of India. Efficacy of a synthetic plant-preferred codon-optimized vip3Aa44 gene was determined towards Helicoverpa armigera (pod borer, cotton bollworm) and Spodoptera litura (cotton leaf worm). To improve the resistance of pigeon pea to pod borer, RNAi strategy of host- delivered-artificial microRNA-mediated targeting of acetylcholinesterase1 (HaAce1) and 20- hydroxy ecdysone receptor (EcR) genes of H. armigera, and the Bt strategy of over expression of a chimeric Bt gene, cry1AcF are being carried out. For robust and durable resistance against insect pest, a binary construct was also developed where both these strategies are being combined. To understand the mechanism of pod borer resistance, wild relatives of pigeon pea are also being analyzed through multiomics approaches. GWAS analysis of a set of 142 pigeon pea genotypes for various yield related traits, especially related to flowering and pod/seeds etc., led to identification of about 20 SNPs. Co-integration of these SNPs with the in-house transcriptome data identified two robust SNPs for days to flowering and fifty per cent flowering across three seasons, which were positioned on a 5 kb unanchored scaffold. This region codes for a novel but uncharacterized protein. A set of 100 pigeon pea genotypes were screened for various seed quality parameters including protein, iron and zinc content and contrasting genotypes for these parameters were identified. To confer aphid resistance in Indian mustard, transgenic plants using either RNAi or protease inhibitors and plant lectins over-expression-based strategies, have been developed. Cystatin from pigeon pea genome has been found to cause ~72 per cent mortality of Aphids. For deriving trait associated molecular markers, potential genomic regions and differentially expressed genes in alien introgression based Alternaria resistant lines in Indian mustard have been identified. Arabidopsis transgenic over-expressing WRKY33 gene have been developed for analysis of Alternaria resistance. Based on pathotyping of 10 isolates of Albugo candida, cv. GSL-1 of B. napus and EC206642, an accession of B. carinata, were found to be immune to all the ten isolates. A non-injury method of disease inoculation has been standardized for screening against Sclerotinia stem rot infection in B. juncea. Based on screening three tolerant genotypes, RH1222-28 of B. juncea, B. nap114 of B. napus and Bcar115 of B. carinata were identified. The whole-genome sequencing of an Indian isolate ‘ESR-01’ of S. sclerotiorum was completed. Constructs for genome editing of Cytokinin oxidase/dehydrogenase (CKX) were developed. Protoplast isolation and transformation technique in B. juncea has been standardized. An efficient regeneration protocol for B. oleracea var. botrytis cv. Pusa iv Meghna has been developed using hypocotyls as explants. Stability analysis of resynthesized B. juncea lines (RBJ 1 to RBJ-92) was carried out. Genome size of wild mustard species was estimated through flow cytometry. Genome size of Orobanchea egyptica, a total root parasite of Brassica was also was estimated and found to be around 3900 Mbp, which is much larger than its host. Apart from the above significant research achievements, ICAR-NIPB is also actively involved in organizing various events for the benefit of farmers. Twenty-nine Kisan Goshthis and 330 demonstrations were conducted in the six adopted villages of Utter Pradesh under the flagship Mera Gaon Mera Gaurav (MGMG) initiative of Government of India during the year. Several trainings for farmers were held and seeds of superior varieties of rice, mustard, pigeon pea and wheat were distributed to farmers under Scheduled Caste Sub-Plan (SCSP) program. The institute also showcased its products and activities in the Kisan mela organized by ICAR-IARI. Human resource development in the area of plant biotechnology is one of the mandates of our institute. Institute undertakes postgraduate teaching in the Molecular Biology and Biotechnology discipline of ICAR-IARI. Currently 28 Ph.D. and 17 M.Sc. students are registered in this discipline in our institute. In the period under report, seven Ph.D. and five M.Sc. students were awarded degrees by ICAR-IARI. The training programs conducted for students and scientific and teaching faculty of the NARS system