High transformation and gene editing efficiencies in sorghum-produced, transgene-free SDN1-edited plants exhibit precise mutations, reduced germination stimulants, and enhanced resistance to Striga infection. Sorghum (Sorghum bicolor L.) is a primary food staple grain for millions in Sub-Saharan Africa (SSA). It is mainly constrained by the parasitic weed Striga, which causes up to 100

Pearl millet is a nutritious and climate-resilient cereal extensively grown in the arid and semi-arid regions of South Asia and Sub-Saharan Africa. Despite its exceptional nutritional qualities, pearl millet suffers from limited acceptance, partly due to the short shelf life of its milled flour. The stored flour quickly becomes rancid after 7-10 days due to the rancidity of fatty acids and oils caused by various factors, including enzymes and metal ions. In this study, we examined high iron (biofortified [ICMH-1202 (BF5) and Dhanashakti (BF6)] and regular [PA-9285 (NBF2) and MP-7872 (NBF4)] millet grain-based stored flour. Hydrolysis of triacylglycerols led to the accumulation of free fatty acids. Data on acid value, peroxide value, and enzymatic activities showed that high iron lines exhibit a higher rate of lipid oxidation and peroxidation after 45 days of storage. The biochemical profiling of these lines indicated that the high-iron and low-iron lines are comparable regarding rancidity-linked traits until 14 days after grinding for most surrogate traits. These findings suggest that iron-rich flour is likely more susceptible to rancidity than low-iron lines after 45 days of storage. The results indicate that high-iron varieties would not affect the general Indian practices of using flour within 14 days, but they may require post-harvest stabilization to enhance shelf life beyond two weeks. However, it is crucial to emphasize that iron-rich varieties are vital in addressing hunger and malnutrition. These exploratory results suggest that heat treatment could help improve the shelf life of iron-rich pearl millet grains on a smaller scale, but a better solution is still needed for large-scale commercialization and to monitor this key trait in released varieties without compromising their nutritional content.

Rising atmospheric CO2 significantly impacts crop productivity and nutritional quality, posing challenges to global food security. Pearl millet (Pennisetum glaucum (L.) R. Br.), a climate resilient nutri-cereal, plays a vital role in food and nutrition security particularly in arid and semi-arid regions of India and sub-Saharan African countries. However, its response to changing climate conditions such as elevated CO2 are not well known. This study assessed the response of various pearl millet genotypes, including hybrids and inbred lines to elevated CO2 (550 and 700 ppm) from the current level of 420 ppm. Elevated CO2 resulted in enhanced plant height, chlorophyll content, and nitrogen balance index. However, average grain yield recorded 1.2 % reduction at 550 ppm and 28.8 % at 700 ppm. Flavonoid concentration increased at 550 ppm (5.1 %) but decreased at 700 ppm (14.5 %). Average grain Fe and Zn content increased at 550 ppm by 4.25 % and 6.12 %, respectively but declined at 700 ppm by 4.01 % and 7.04 %; however in ICMB 92111, ICMB 92888, HHB67Imp and NBH 4903 increased Fe accumulation was recorded at 700 ppm) Grain protein content decreased significantly (1.12 % at 550 ppm, 13.4 % at 700 ppm), while fodder protein increased (16.01 % at 550 ppm, 15.19 % at 700 ppm). These findings highlight the complex effects of CO2 fertilization on pearl millet's productivity and nutritional profile; the crop remains relatively resilient up to 500 ppm CO2 but becomes more susceptible to negative impacts at 700 ppm. Therefore, large-scale germplasm evaluation and targeted breeding efforts are essential to develop climate-resilient genotypes with stable yields and enhanced nutrient content under future CO2 conditions.

Cytokinins (CKs) are multifaceted hormones that regulate growth, development, and stress responses in plants. Cytokinins have been implicated in improved panicle architecture and grain yield however are inactivated by the enzyme cytokinin oxidase (CKX). In this study we developed a cytokininn oxidase2 ( Osckx2 ) deficient mutant using CRISPR/Cas9 gene editing in indica rice and assesses its function under water deficit and salinity conditions. Loss of OsCKX2 function increased grain number, secondary panicle branching and overall grain yield through improved cytokinin content in the panicle tissue. Under drought conditions, the Osckx2 mutant conserved more water and demonstrated improved water-saving traits. Through reduced transpiration, Osckx2 mutants showed improved survival response than wild type to unset dehydration stress. Further, Osckx2 maintained chloroplast and membrane integrity and showed significantly improved photosynthetic function under drought conditions through enhanced antioxidant protection systems. The OsCKX2 function negatively affects panicle grain number and drought tolerance, with no discernible impact in response to salinity. The finding suggests the utility of the beneficial Osckx2 allele in breeding to develop climate-resilient high-yielding cultivars for future food security.

In India, pigeonpea is a drought-tolerant crop that is susceptible to more than 100 diseases, including viruses, bacteria, nematodes, fungus, and phytoplasma. The restrictions of utilising chemical fungicides make managing soil-born diseases, like Fusarial wilts, difficult. While using resistant cultivars is preferable, the emergence of novel disease variations frequently poses a challenge. The objectives of this study are to investigate root proteome responses, discover host plant proteins for cultivars that are resistant, and comprehend the molecular mechanisms behind the interactions between F. udum and pigeonpea. Proteomics was used to examine genotypes of pigeonpea fusarium wilt-susceptible (ICP 2376) and resistant (ICP 9174). To make ingress easier, seedlings were removed, cleaned, and chopped off. Both genotypes underwent total protein extraction, and the pigeonpea root protein underwent two-dimensional gel electrophoresis. A combination search of PMF and MS/MS data against the taxonomy of Viridiplantae was used to identify proteins. The goal is to comprehend the molecular processes that control plant resistance and susceptibility. Twelve of the 141 differentially expressed protein locations in the interaction between F. udum and pigeonpea were satisfactorily characterised. Based on their biological roles, seven differentially expressed proteins were found in the resistant cultivar and categorised into seven functional groups. Proteome profiles provided insight into the defense mechanism of pigeonpea against Fusarium udum infection by identifying two proteins, R56 and S41, as components of the defense mechanism. Greater accumulation of R56 in resistant cultivars suggests F. udum-induced defense, whereas down-regulation of S41 in susceptible cultivars supports direct defense.

Restoration factors (Rfs) belonging to the pentatricopeptide repeat proteins (PPRs) family play an essential role in plant growth and development including their binding to CMS-associated mitochondrial RNAs leading to fertility restoration. The present study identified 22 mitochondrial-specific PPRs in pigeonpea and explored the underlying mechanisms of restoration of fertility in the A4 CMS system through yeast-three hybrid studies. The identified gene was functionally validated through transgenic expression in Arabidopsis model system and obtained conclusive evidence that the identified Rf-PPR was responsible for fertility restoration. The sub-cellular localization studies implied that the identified Rf-PPR is mitochondrial targeting. The study demonstrated that due to the interaction between mitochondrial CMS mRNA and nuclear Rf-PPR protein, post-transcriptional modification occurred, leading to the inability to translate and accumulate cytotoxic CMS protein resulting in fertility restoration. The study specifically looks into the RNA-protein interaction occurring at the nucleo-cytoplasmic level in the A4 cytoplasm of Cajanus cajanifolius .Highlights The study identifies the restoration of fertility genes corresponding to the CMS-causing orf147 gene.### Competing Interest StatementThe authors have declared no competing interest.* CMS : Cytoplasmic male sterility Col-0 : Columbia-0 DDO/X/A : Double dropout media / X-α-gal/ Aureobasidin A GFP : Green fluorescent protein qRT-PCR : quantitative real-time PCR PPR : Pentatricopeptide repeat protein Rf : Restoration factors WT : Wild type Y3H : Yeast three hybrid system

A comprehensive understanding of the nucleocytoplasmic interactions that occur between genes related to the restoration of fertility and cytoplasmic male sterility (CMS) provides insight into the development of hybrids of important crop species. Modern biotechnological techniques allow this to be achieved in an efficient and quick manner. Heterosis is paramount for increasing the yield and quality of a crop. The development of hybrids for achieving heterosis has been well-studied and proven to be robust and efficient. Cytoplasmic male sterility (CMS) has been explored extensively in the production of hybrids. The underlying mechanisms of CMS include the role of cytotoxic proteins, PCD of tapetal cells, and improper RNA editing of restoration factors. On the other hand, the restoration of fertility is caused by the presence of restorer-of-fertility (Rf) genes or restorer genes, which inhibit the effects of sterility-causing genes. The interaction between mitochondria and the nuclear genome is crucial for several regulatory pathways, as observed in the CMS–Rf system and occurs at the genomic, transcriptional, post-transcriptional, translational, and post-translational levels. These CMS–Rf mechanisms have been validated in several crop systems. This review aims to summarize the nucleo-mitochondrial interaction mechanism of the CMS–Rf system. It also sheds light on biotechnological interventions, such as genetic engineering and genome editing, to achieve CMS-based hybrids.

Advances in biocontrol potentials and fungicide resistance are highly desirable for Trichoderma. Thus, it is profitable to use mutagenic agents to develop superior strains with enhanced biocontrol properties and fungicide tolerance in Trichoderma. This study investigates the N-methyl-n-nitro-N-nitrosoguanidine (NTG) (100 mg/L) induced mutants of Trichoderma asperellum. Six NTG (3 each from 1st & 2nd round) induced mutants were developed and evaluated their biocontrol activities and carbendazim tolerance. Among the mutant N2-3, N2-1, N1 and N2-2 gave the best antagonistic and volatile metabolite activities on inhibition of chickpea F. oxysporum f. sp. ciceri, B. cinerea and R. bataticola mycelium under in vitro condition. Mutant N2-2 (5626.40 μg/ml) showed the highest EC50 value against carbendazim followed by N2-3 (206.36 μg/ml) and N2-1 (16.41 μg/ml); and succeeded to sporulate even at 2000 μg/ml of carbendazim. The biocontrol activity of N2-2 and N2 with half-dose of carbendazim was evaluated on chickpea dry root rot under controlled environment. Disease reduction and progress of the dry root rot was extremely low in T7 (N2-2 + with half-dose of carbendazim) treatment. Further, carbendazim resistant mutants demonstrated mutation in tub2 gene of β-tubulin family which was suggested through the 37 and 183 residue changes in the superimposed protein structures encoded by tub2 gene in N2 and N2-2 with WT respectively. This study conclusively implies that the enhanced carbendazim tolerance in N2-2 mutant did not affect the mycoparasitism and plant growth activity of Trichoderma. These mutants were as good as the wild-type with respect to all inherent attributes.

Orf147, a cytotoxic peptide, has been found to cause cytoplasmic male sterility (CMS) in Cajanus cajanifolius (pigeonpea). In our study, Orf147 was introduced into self-pollinating Cicer arietinum (chickpea) using Agrobacterium-mediated transformation for induction of CMS. The stable integration and expression of the transgene has been assessed through PCR and qRT-PCR analysis. In addition, phenotypic sterility analysis has been performed, considering developmental parameters like flower development, pod formation and flower drop. Transgene inheritance analysis demonstrates that out of the five PCR positive events in the T0 generation, two events have segregated according to the Mendelian segregation ratio (3:1) in the T2 generation. Further, pollen viability test using microscopic analysis confirms the induction of partial CMS in transgenic chickpea. The study holds significant value regarding the heterosis of self-pollinating legumes like chickpea. As a part of the prospect, exploring inducible promoters of species-specific or related legumes would be the next step to developing a two-line hybrid system.

Aflatoxins are immunosuppressive and carcinogenic secondary metabolites, produced by the filamentous ascomycete Aspergillus flavus, that are hazardous to animal and human health. In this study, we show that multiplexed host-induced gene silencing (HIGS) of Aspergillus flavus genes essential for fungal sporulation and aflatoxin production (nsdC, veA, aflR, and aflM) confers enhanced resistance to Aspergillus infection and aflatoxin contamination in groundnut (<20 ppb). Comparative proteomic analysis of contrasting groundnut genotypes (WT and near-isogenic HIGS lines) supported a better understanding of the molecular processes underlying the induced resistance and identified several groundnut metabolites that might play a significant role in resistance to Aspergillus infection and aflatoxin contamination. Fungal differentiation and pathogenicity proteins, including calmodulin, transcriptional activator-HacA, kynurenine 3-monooxygenase 2, VeA, VelC, and several aflatoxin pathway biosynthetic enzymes, were downregulated in Aspergillus infecting the HIGS lines. Additionally, in the resistant HIGS lines, a number of host resistance proteins associated with fatty acid metabolism were strongly induced, including phosphatidylinositol phosphate kinase, lysophosphatidic acyltransferase-5, palmitoyl-monogalactosyldiacylglycerol Δ-7 desaturase, ceramide kinase-related protein, sphingolipid Δ-8 desaturase, and phospholipase-D. Combined, this knowledge can be used for groundnut pre-breeding and breeding programs to provide a safe and secure food supply.

Technologies and innovations are critical for addressing the future food system needs where genetic resources are an essential component of the change process. Advanced breeding tools like “genome editing” are vital for modernizing crop breeding to provide game-changing solutions to some of the “must needed” traits in agriculture. CRISPR/Cas-based tools have been rapidly repurposed for editing applications based on their improved efficiency, specificity and reduced off-target effects. Additionally, precise gene-editing tools such as base editing, prime editing, and multiplexing provide precision in stacking of multiple traits in an elite variety, and facilitating specific and targeted crop improvement. This has helped in advancing research and delivery of products in a short time span, thereby enhancing the rate of genetic gains. A special focus has been on food security in the drylands through crops including millets, teff, fonio, quinoa, Bambara groundnut, pigeonpea and cassava. While these crops contribute significantly to the agricultural economy and resilience of the dryland, improvement of several traits including increased stress tolerance, nutritional value, and yields are urgently required. Although CRISPR has potential to deliver disruptive innovations, prioritization of traits should consider breeding product profiles and market segments for designing and accelerating delivery of locally adapted and preferred crop varieties for the drylands. In this context, the scope of regulatory environment has been stated, implying the dire impacts of unreasonable scrutiny of genome-edited plants on the evolution and progress of much-needed technological advances.

Pearl millet [Pennisetum glaucum (L) R. Br.] is an important cereal crop of the semiarid tropics, which can withstand prolonged drought and heat stress. Considering an active involvement of the aquaporin (AQP) genes in water transport and desiccation tolerance besides several basic functions, their potential role in abiotic stress tolerance was systematically characterized and functionally validated. A total of 34 AQP genes from P. glaucum were identified and categorized into four subfamilies, viz., plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), nodulin-26-like intrinsic proteins (NIPs), and small basic intrinsic proteins (SIPs). Sequence analysis revealed that PgAQPs have conserved characters of AQP genes with a closer relationship to sorghum. The PgAQPs were expressed differentially under high vapor pressure deficit (VPD) and progressive drought stresses where the PgPIP2;6 gene showed significant expression under high VPD and drought stress. Transgenic tobacco plants were developed by heterologous expression of the PgPIP2;6 gene and functionally characterized under different abiotic stresses to further unravel their role. Transgenic tobacco plants in the T2 generations displayed restricted transpiration and low root exudation rates in low- and high-VPD conditions. Under progressive drought stress, wild-type (WT) plants showed a quick or faster decline of soil moisture than transgenics. While under heat stress, PgPIP2;6 transgenics showed better adaptation to heat (40°C) with high canopy temperature depression (CTD) and low transpiration; under low-temperature stress, they displayed lower transpiration than their non-transgenic counterparts. Cumulatively, lower transpiration rate (Tr), low root exudation rate, declined transpiration, elevated CTD, and lower transpiration indicate that PgPIP2;6 plays a role under abiotic stress tolerance. Since the PgPIP2;6 transgenic plants exhibited better adaptation against major abiotic stresses such as drought, high VPD, heat, and cold stresses by virtue of enhanced transpiration efficiency, it has the potential to engineer abiotic stress tolerance for sustained growth and productivity of crops.

ABSTRACT Commercialization and utilization of pearl millet ( Pennisetum glaucum L.) by consumers and processing industry is constrained due to rapid onset of rancidity in its milled flour. We studied the underlying biochemical and molecular mechanisms to flour rancidity in contrasting inbreds under 21-day accelerated storage. Rapid TAG decrease was accompanied by FFA increase in high rancidity genotype compared to the low rancidity line, that maintained lower FFA and high TAG levels, besides lower headspace aldehydes. DNA sequence polymorphisms observed in two lipase genes revealed loss-of-function mutations that were functionally confirmed in yeast system. We outline a direct mechanism for mutations in these key TAG lipases in pearl millet and the protection of TAG and fatty acids from hydrolytic and oxidative rancidity respectively,. Natural variation in the PgTAGLip1 and PgTAGLip2 genes may be selected through marker assisted breeding or by precision genetics methods to develop hybrids with improved flour shelf life.

Defensins are broad-spectrum antimicrobial peptides that play an important role in providing innate immunity to various biotic stresses in plants. We identified and characterized 22 defensin (DEF) and defensin-like (DEFL) genes in chickpea ( Cicer arientinum ) based on their structures, expression, chromosomal localization, conserved motifs, and cis -regulatory elements. The localization of DEF and DEFL genes in chickpea genome revealed the presence of at least two clusters that are likely evolved through local gene duplications. Genotype-specific responses of several CaDEF and CaDEFL genes in fungal bioassays suggested their involvement in defense against fungal pathogens such as hemi-biotrophic F. oxysporum f. sp. ciceris and dry root rot causing necrotrophic R. bataticola . Molecular docking studies revealed interactions of CaDEFs with fungal plasma membrane components such as phosphatidylserine (PS) and glucosylceramide (GluCer) and their binding sites were identified. Our data will be useful to identify potential candidate genes and their role in host-plant resistance in chickpea, besides presenting opportunities for their potential for possible deployment in other crops.

Pearl millet is an important cereal crop of semi-arid regions since it is highly nutritious and climate resilient. However, pearl millet is underutilized commercially due to the rapid onset of hydrolytic rancidity of seed lipids post-milling. We investigated the underlying biochemical and molecular mechanisms of rancidity development in the flour from contrasting inbred lines under accelerated aging conditions. The breakdown of storage lipids (triacylglycerols; TAG) was accompanied by free fatty acid accumulation over the time course for all lines. The high rancidity lines had the highest amount of FFA by day 21, suggesting that TAG lipases may be the cause of rancidity. Additionally, the high rancidity lines manifested substantial amounts of volatile aldehyde compounds, which are characteristic products of lipid oxidation. Lipases with expression in seed post-milling were sequenced from low and high rancidity lines. Polymorphisms were identified in two TAG lipase genes (PgTAGLip1 and PgTAGLip2) from the low rancidity line. Expression in a yeast model system confirmed these mutants were non-functional. We provide a direct mechanism to alleviate rancidity in pearl millet flour by identifying mutations in key TAG lipase genes that are associated with low rancidity. These genetic variations can be exploited through molecular breeding or precision genome technologies to develop elite pearl millet cultivars with improved flour shelf life.

Late embryogenesis abundant (LEA) genes display distinct functions in response to abiotic stresses in plants. In pearl millet (Pennisetum glaucum L.), a total of 21 PgLEA genes were identified and classified into six groups including LEA1, LEA2, LEA3, LEA5, LEA7, and dehydrins (DHN). Open reading frames (ORFs) of PgLEAs range from 291 bp (PgLEA1-1) to 945 bp (PgLEA2-11) and distributed randomly among the seven chromosomes. Phylogenetic analysis revealed that all PgLEA proteins are closely related to sorghum LEA proteins. The PgLEAs were found to be expressed differentially under high progressive vapor pressure deficit (VPD), PgLEA7 was significantly expressed under high VPD and was selected for functional validation. In silico analysis of the PgLEA promoter regions revealed abiotic stress-specific cis-acting elements such as ABRE, CCAAT, MYBS, and LTRE. Based on the type of motifs, PgLEAPC promoter (758 bp), its deletion 1 (PgLpd1, 349 bp) and deletion 2 (PgLpd2, 125 bp) were cloned into the plant expression vector pMDC164 having the promoter-less uidA gene. All the three plant expression vectors were introduced into tobacco through Agrobacterium tumefaciens-mediated transformation to obtain T-1 and T-2 generations of transgenic plants. Based on expression of the uidA gene, tissue-specific expression was observed in mature stems, roots and seedlings of PgLEAPC and PgLpd1 carrying transgenics only. While the transgenic PgLEAPC plants displayed significantly higher uidA expression in the stem and root tissues under salt, drought, heat, and cold stresses, very low or no expression was observed in PgLpd1 and PgLpd2 transgenics under the tested stress conditions. The results of this study indicate that the complete promoter of PgLEAPC plays a role in developing abiotic stress tolerance in plants.

Pearl millet (Pennisetum glaucum [L.] R. Br.) is an important crop capable of growing in harsh and marginal environments, with the highest degree of tolerance to drought and heat stresses among cereals. Diverse germplasm of pearl millet shows a significant phenotypic variation in response to abiotic stresses, making it a unique model to study the mechanisms responsible for stress mitigation. The present study focuses on identifying the physiological response of two pearl millet high-resolution cross (HRC) genotypes, ICMR 1122 and ICMR 1152, in response to low and high vapor pressure deficit (VPD). Under high VPD conditions, ICMR 1152 exhibited a lower transpiration rate (Tr), higher transpiration efficiency, and lower root sap exudation than ICMR 1122. Further, Pg-miRNAs expressed in the contrasting genotypes under low and high VPD conditions were identified by deep sequencing analysis. A total of 116 known and 61 novel Pg-miRNAs were identified from ICMR 1152, while 26 known and six novel Pg-miRNAs were identified from ICMR 1122 genotypes, respectively. While Pg-miR165, 168, 170, and 319 families exhibited significant differential expression under low and high VPD conditions in both genotypes, ICMR 1152 showed abundant expression of Pg-miR167, Pg-miR172, Pg-miR396 Pg-miR399, Pg-miR862, Pg-miR868, Pg-miR950, Pg-miR5054, and Pg-miR7527 indicating their direct and indirect role in root physiology and abiotic stress responses. Drought responsive Pg-miRNA targets showed upregulation in response to high VPD stress, further narrowing down the miRNAs involved in regulation of drought tolerance in pearl millet.

For millennia, natural and artificial selection has combined favourable alleles for desirable traits in crop species. While modern plant breeding has achieved steady increases in crop yields over the last century, on the current trajectory we will simply not meet demand by 2045. Novel breeding strategies and sources of genetic variation will be required to sustainably fill predicted yield gaps and meet new consumer preferences. Here, we highlight that stepping up to meet this grand challenge will increasingly require thinking 'beyond the gene'. Significant progress has been made in understanding the contributions of both epigenetic variation and cis-regulatory variation to plant traits. This non-genic variation has great potential in future breeding, synthetic biology and biotechnology applications.

Aflatoxigenic fungi infect various agricultural commodities and produce a class I carcinogen aflatoxin B1 (AFB1). Sixteen bacterial isolates were screened from idli batter and other fermented foods, for antagonistic effect against Aspergillus spp. Based on the preliminary observation, three bacterial isolates from idli batter namely, IB1, IB4, IB7, and one from mango wine namely, MW2 were selected and further characterized. IB1 was found to be closely related to Lactobacillus plantarum argentoratensis while IB4, IB7, and MW2 were closely related to Enterococcus faecium according to their 16sRNA sequencing. Invitro and invivo antifungal tests in peanut revealed that cell free supernatant (CFS) obtained from these isolates inhibited fungal growth and significantly suppressed AFB1 production, and expression of aflD, aflP, OrdB and aflR genes. Further, microscopic examination showed that all isolates could reduce the conidial development and inhibits spore formation. The highest antifungal activity was observed in CFS of L. plantarum MN880325 and E. faecium MN880376, with an inhibition zone of >= 28 mm in PDA and >= 97% reduction in aflatoxin production on peanut kernels. ESI-MS and NMR spectroscopy revealed the active compounds from L. plantarum MN880325 and E. faecium MN880376 to be 3-phenyllactic acid and 4-hydroxyphenyllactic acid respectively.