Nanaji Deshmukh Plant Phenomics Centre, ICAR-IARI, New Delhi: A State of Art Facility for Research for Combating Climate Change

Basic and Cutting Edge Technology Research

Structural Genomics (Genome Sequencing) of Crop Plants to Identify and Utilize Novel Genes, Pathways, and Metabolites

ICAR successfully carried out genome sequencing to decode the structural genomic information of multiple crop plants and microbial strains in collaboration with other institutes or solely by NIPB scientists. The important crops that are sequenced in collaboration are rice, wheat, tomato, mango, flax, jute, tea, guar while pigeonpea genome was sequenced exclusively by NIPB scientists.
In addition to deciphering crop plant genomes, NIPB scientists also decoded the structural genome of a rice pathogen Magnaporthe oryzae Xanthomonas oryzae and wheat pathogen Puccinia spp to understand the genomic variation and identify candidate avirulence (Avr) genes. 

Different plant genomes sequenced by ICAR-NIPB for identification and utilization of novel genes, pathways, and metabolites

 

Isolation, Cloning and Functional Validation of Pi54 Gene for Resistance to Rice Blast

The Pi54(Pi-kh) gene conferring a high degree of resistance to diverse strains of the fungus Magnaporthe oryzae was mapped,cloned and functionally validated in 2005. In an effort to identify more effective forms of this gene, two orthologues of Pi54 named as Pi54rh. and Pi54 of isolated from the blast-resistant wild species of rice, Oryza rhizomatis, and O.officinalis, respectively using allele mining approach and validated by gene complementation. The major blast resistance gene Pi54 has been transferred to many rice varieties using marker assisted selection by the breeders in India and abroad.

Pi54rhtransgenic lines impart resistance to M. oryzae isolates. Disease reaction (0–5 scale)of transgenic line derived from TP 8.3.45.2 vis-à-vis susceptible TP309 against six isolates of M. oryzae(a). Disease reaction of transgenic lines with six fungal isolates (Mawana, MG79, MSF9, RML29, Shihunta1and Deh-1) collected from different geographical regions of India, NT:non-transgenic lines,T: transgenic lines.

 

Transgenic Chickpea (Cicerarietinum L.) for Pod Borer Resistance

Chickpea cv. DCP 92-3 was genetically transformed using Agrobacterium tumefaciens strain EHA105, harboring binary vector p BinAR containing cry1Aabc gene and nptII as selectable marker. Multiple shoots were induced and elongated from meristematic region, co-cultivated with Agrobacterium, under the influence of different growth regulators The transgenicity of the regenerants was confirmed for presence and expression of transgene by molecular analyses such as PCR, Southern blot, strip test, ELISA (Qualitative and Quantitative), western blot and RT-PCR. Trait efficacy of the lines was assessed using detached leaf bioassay, pod bioassay and whole plant bioassay using 2 d old larvae of the gram pod borer (Helicoverpaarmigera H.). Performance of two events (IPCa2 and IPCa4) was assessed under field conditions in event selection trial.

Detached leaf and pod bioassay using 2 d old gram pod borer larvae.

 

Transgenic Pigeonpea for Pod Borer Resistance

Bt transgenic pigeonpea carrying insecticidal crystalline proteins (ICPs),cry1AcF (a chimeric gene with cry1Ac and cry1F domains) and cry2Aaweredeveloped and evaluated under controlled glass house condition. After stringent molecular and bioefficacy analyses in advanced generations identified 8 superior events were identified and RCGM clearance has been obtained. Currently, we are processing for NOC from state government.

Transgenic pigeonpea resistant to pod borer and bioefficacy analysis of the transgenic plants under nethouse conditions

 

Transgenic Sorghum with Cry1B Gene for Shoot Borer Resistance

Transgenics in sorghum to mitigate the shoot borer were developed by deploying synthetic cry1B gene driven by maize ubiquitin promoter using M35-1 genotype through Agrobacterium-mediated transformation. The developed transgenics were characterized for the insertion and integration of cry1B gene through PCR and Southern analysis. Further, the expression of cry1B gene was quantified through ELISA and Western analysis. The transgenic event NRCSCRY1B (event-19), identified and evaluated through insect bioassay studies through leaf disc and whole plant bioassays showed resistance to stem borer. The same event was further evaluated under open field trial (BRL-1) and the data generated indicated that the sorghum transgenic plants with cry1B gene showed resistance against stem borer.

Confined field trial of sorghum transgenic against stem borer:
(a) artificial infestation of stem borer larvae using bazooka during field trial;
(b) Stem tunnelling in infested control plants; Resistant transgenic line
(c) resistant transgenic line;
(d) susceptible control line.

 

Transgenic Tomato for Fruit Borer Resistance

The cry1Ac gene was introduced into an improved inbred line of tomato (KashiVishesh) using Agrobacterium mediated transformation. Presence of the transgene was confirmed by PCR amplification, and single locus insertion and stability of the transgene was confirmed by segregation analysis of T1 seeds from T0 plants. Sixteen independent transgenic events showed single copy cry1Ac gene integration. Homozygous T5 plants of eight cry1Ac transgenic events (VRTT–1 to VRTT–8) were evaluated under event selection trial. Insect bioassay by feeding second-instar TFB larva on fruits of transformed and non- transformed control plants showed that the event VRTT–5 had the highest mortality and weight loss after 9 days of inoculation.

Cry1Ac transgenic tomato events in containment condition.

 

Transgenic Tomato for Drought Tolerance

Drought stress tolerant tomato (cv. KashiVishesh), containing BcZAT12was developed and presence of the transgene was confirmed by PCR amplification in 17 independent events. BcZAT12 transgenic lines, along with drought tolerant genotypes (H-88-78-1 and VRT-32-1), were screened for different biochemical indicate that the BcZAT12 lines are better in comparison to drought tolerant genotypes. Biochemical estimation at 40% FC indicate that BcZAT12 lines were much superior to the corresponding drought tolerant genotypes.

BcZAT12 transgenic tomato events tolerant to abiotic stress. Transgenic events in the containment facility.

 

Transgenic Brinjal for Resistance to Fruit and Shoot Borer

An improved inbred line of eggplant (IVBL-9) was transformed with cry1Ac gene. Nine events (IVBE-1 to IVBE-9) of Cry1Ac transgenic brinjal (IVBL-9) were evaluated under event selection trial. Based on Cry1Ac expression in different tissues and mortality of larva fed on brinjal fruitsIVBE-8 event was selected as the best event(Fig. 9).

BT (cry1Ac) transgenic brinjal events resistant to fruit and shoot borer.

 

Development of DNA Chip for Finger-printing and Rice Breeding

 

Genome Editing of Rice

• Rice DST (DROUGHT AND SALT TOLERANCE) gene, encoding a Zinc finger transcription factor, is a negative regulator of drought and salt tolerance and yield. CRISPR-Cas9 SDN1 genome editing method was employed to create loss of function mutants of DST gene in rice cv. MTU1010.

• Transgene free homozygous dst mutants showed reduced stomatal density and water use per unit leaf area, and enhanced tiller number, leaf area, grains per panicle and grain yield under non-stress conditions.

• The dst mutants (Δ366bp,Δ2bpandΔ24bp are dst mutants with 366, 2 and 24 bp deletions, respectively) exhibited enhanced tolerance to salt (200 mM) and drought (-70 to -90 KPa) stresses, and produced significantly higher grain yield than WT after recovery.

 

Herbicide Tolerant (HT) Mutant of Nagina 22

• Screening of EMS-induced 100,000 M2 mutants of Nagina 22 led to the identification of herbicide "Imazethapyr" tolerant mutant “Robin”. Molecular characterization revealed that the mutation in Acetohydroxy Acid Synthase (AHAS) gene confers herbicide tolerance. The mutant allele is named AHASrb.

• By using maker assisted introgression, AHASrb allele was introgressed in to India’s leading basmati rice varieties Pusa Basmati 1121 and Pusa Basmati 1509 to develop herbicide tolerant Pusa Basmati 1979 and Pusa Basmati 1985.

• These varieties suitable for Direct Seeded Rice (DSR) cultivationsave water and Rs. 1250/- per hectare in weed control as compared with conventional varieties.

 

Drosophila Wingless Mutant

• The wingless mutant of Drosophila was isolated from EMS mutant population at Division of Genetics, IARI (Sharma and Chopra. 1976.Dev Biol. 48:461-5).It lacks one or both wings and is a hypomorphic allele of wingless gene.

• Scientist discovered the gene int-1 (integration site-1) while studying occurrence mammary tumors in mice after infection of Mammary Tumor Virus (MMTV). Subsequent studies identified int-1 is homologous to Drosophila segment polarity gene wingless. Scientists identified many genes across animal kingdom similar to wingless gene of Drosophila.

• Later from Wingless and int-1, the name Wnt was given to this gene family.