Development of embryo rescue protocol: In-vitro culture of cotton ovules (Embryo rescue technique) was employed to get F1 hybrid between distant species i.e., G. hirsutum and G. arboreum. A new embryo culture protocol is valuable for inter-specific (diploid × tetraploid) hybridization and rapid generation advancement for introgression breeding has been standardized.
Standardization of somatic embryogenesis mediated regeneration protocol: A tissue culture media regime has been standardized for Somatic Embryogenesis mediated regeneration in Coker 310 and Coker 312 genotypes.
In-planta transformation protocol in cotton: Sonication and vacuum infiltration assisted transformation of transgene was standardized in planta. Transgene delivery was assayed and validated using transient gusassay.
Transgenic cotton development
Next generation transgenic Cotton: ICAR-CICR is putting dedicated efforts to develop next-generation transgenic cotton varieties through introgression and pyramiding of some of the transgenic events Tg2E13 (cry1Ac gene),CH12 (cry2Ax1 gene), UASD No. 78 (cry1Ac gene) developed by Indian Public sector Institutes for durable pest management and sustainable cotton production.
Protocol for detection of transgene from matured fibers samples of cotton: A protocol has been standardized for PCR based detection of the presence/absence of GM-events/ transgene from immature fiber.
Bt Referral Laboratory, ICAR-CICR, Nagpur: Under subsection 1 of section 4 of Seeds Act 1966, Central Government declared ICAR-CICR, Nagpur as the Referral Lab for Bt testing. Accordingly, the designated laboratory tests referred samples from Court, State Government and other agencies. In addition, Bt Referral Laboratory provides services to AICRP on Cotton in evaluation and release of Bt varieties and hybrids for their commercial cultivation. Cotton samples are also tested here for presence of BT and HT cotton events and monitor illegal cultivation of unapproved HT Bt cotton.
PCR based diagnostic protocol for HT/BT events: Gene/event specific primers (cp4epsps, MON1445 and MON88913 (Roundup Ready-Flex)) were designed for detection presence of herbicide tolerant gene(s) and event(s) and illegal cultivation of HT cotton in Maharashtra. The protocol is has been standardized for detection of willful and non-willful contamination of samples.
Genes for useful traits
Molecular characterization and validation of fiber strength genes: Selected candidate genes (SusA1, GhcesA1 GhcesA2, GhcesA7, Ghcobl4 and GhLIM) involved in fibre development has been validated, particularly for their association withfibre strength trait.
Genome-wide identification of LIM genes in Asiatic cotton (Gossypium arboreum):
Genome-wide analysis of LIM gene family in desi cotton was carried out which identified twenty LIM domain proteins (GaLIMs). Those twenty GaLIMs showed variable spatiotemporal expression patterns. Based upon biotic and abiotic stress response studies, we have identified GaDA2 as a probable candidate gene for stress tolerance in G. arboreum.
Identification of Two Zinc Finger Transcription Factors During Cotton associated with cotton fibre Initiation:
Two Zinc finger Transcription Factors (ZFP5 and ZFP8) were computationally identified from cotton genome database and their gene expression patterns were studied during fiber initiation of Gossypium hirsutum cv MCU5 and its near-isogenic fuzzless-lintless mutant. The study revealed the potential roles of ZFP5 and ZFP8 in cotton fiber initiation.
Molecular mechanism underlying drought tolerance in cotton:
Six Gossypium hirsutum genotypes were studied for drought tolerance, wider adaptability and better fibre quality traits. From this study, it was observed that genotypes IC325280 and LRA5166 showed ABA mediated expression of stress responsive genes and traits. Fourteen drought stress related genes were studied under water stress conditions. The results indicated that both ABA dependent and ABA independent mechanisms of drought tolerance might be operating
differentially in the studied genotypes.
Precision in transfer and editing of target gene is of more significance in genetic manipulation of crop plants. The target gene GhPHYA1 is one of the potential major negative regulators of many genes contributing for cotton fiber quality, boll size, vegetative growth, flowering etc. Therefore, targeted mutagenesis of GhPHYA1 aimed at improvement of useful trait in cotton.
Agrobacterium-mediated genetic transformation of G. hirsutum Coker 312 with CICR- cry2Ab1Ac::chitinase gene constructs and regeneration through somatic embryogenesis found four putative transgenic plants positive for npt-II and chitinase gene with PCR analysis using gene specific primers and 45 putative transgenic plants were regenerated.Unveiling the potential of cotton WNT-like gene in somatic embryogenesis through genetic engineering
Characterization of WNT-like gene/s in plants and functional validation may leads to unveiling their potential role in somatic embryogenesis of cotton. Such an attempt of basic research not only aids for the generation of reproducible somatic embryogenesis protocol but also may create clues for the signalling cascades of WNT-like pathways to uncover other upstream or downstream players in plants.
Putative transgenic callus cultures derived from transformation of Wnt 3A gene cassette in G hirsutum cv. Suraj were confirmed through PCR analysis using combination of gene and vector backbone primers. Isolation, cloning and transformation of Wnt 3A like gene into cotton (Suraj) was attempted for its functional validation through over-expression.Development of elite Bt cotton varieties using potential non-deregulated transgenic events
Introgression and Evaluation of Tg2E13 event: Embryo culture mediated accelerated introgression of Tg2E13 event (crylAc gene) into three genotypes (Suraj, NH615 and CISH3178) was accomplished through backcross breeding under contained facility of ICAR-CICR, Nagpur. To confirm the protein expression, bioefficacy and agronomic performance of the Tg2E13 introgressed plants, a Confined Field Trial was proposed to conduct in the year 2021 -22.
Introgression of CH12 event (cry2Ax1): BC3F,’s of three crosses (Suraj, NH615 and CISH3178) were made, F1’s will be selfed to produce BC3F2. It is proposed to study homozygous plants in BC3F2 generation and shall be further evaluated.
Evaluation of Event UASD 78 (cry1Ac): The event UASD 78 has been characterized and the report was submitted to the Council.
Evaluation of Tma12 (Event 403) on whitefly incidence: Comparative evaluation of Event 403 (Tma-12), non-Event, HS-6 (susceptible genotype) and LPS-141 (resistant genotype) was carried out in 2020-21. Incidence of whitefly on Tma 12 and LPS141 (resistant check) was similar while the incidence on non-Tma 12 plants and HS6 (highly susceptible check) was comparable but significantly higher than in Tma12 and LPS141 genotypes.Exploration of genomic resources for identification of candidate genes and promoters for cotton improvement
The deployment of biotechnology tools for cotton crop improvement necessitates the discovery of novel genes associated with economical important traits and unveiling the possible role of engineered Cry toxin for enhanced toxicity against cotton bollworms. The cry1D nucleotide sequence coding for active toxin was custom synthesized, cloned in bacterial expression vector pET28, expressed in bacterial host E. coli Rosetta garni and BL21 strains.
Recombinant proteins expressed in E. coli BL21 cells were isolated and utilized for testing the efficacy of target Cry1D protein against P. gossypiella, H. armigera and S. frugiperda through diet incorporation method. The preliminary bioassay results were found promising against pink bollworm P. gossypiella.
Isolation of stress (drought and salt) responsive genes for functional validation: Isolated full length coding sequence of GaDARI (1.5kb) and GhNAC9 (861 bp) from G. arboreum Cv Roja and G. hirsutum respectively.Targeted mutagenesis of ghPHYAI through CRISPR/Cas9 in Cotton
PHYA1 is one of the known major potential negative regulator of PHYA2, PHYB PHYC and PHYE genes and RNAi cotton lines of PHYA1 showed vigorous vegetative growth, flowering and early maturity, higher root mass, higher germination, more flowers and bolls, and increase in fibre staple length.
ghPHTAI Chimeric callus cultures for the presence of sgRNA::Cas9 using vector and sgRNA specific primers was established. This methodology helps to shortlist the putative callus cultures. Agrobacterium mediated transformation of Coker 312 hypocotyls was carried out with sgRNA3GhPHYA1::CRISPR / Cas9 cassettes. Somatic embryogenesis has been achieved from the Coker 312 callus cultures harbouring sgRNA3GhPHYA1::CRISPR/Cas9 cassette.Unraveling the Differential Expressed Proteins (DEP) in cotton genotypes with contrasting resistance to leafhopper and development of the protein biomarkers/functional markers for leafhopper resistance :
Discerning the Differential Expressed Proteins (DEP) towards leafhopper resistance aids in identifying the key peptides involved in resistance. Unravelled peptides sequence information would help in development of functional markers for marker-assisted selection towards leafhopper resistance in cotton.Information Compiled by Dr. Vijay Waghmare, Dr. M. Sabesh, Dr.A.Manivannan Source: CICR Annual reports: updated: 26:04:2023