Tapping into the Potential of Underutilized Niger (Guizotia abyssinica (L. f.) Cass.) Through Breeding and Biotechnological Tools

Abstract

Niger (Guizotia abyssinica (L. f.) Cass.), an oilseed crop from the Compositae family, thrives in temperate and tropical climates. Its small seeds, rich in oil (50–60% biocrude), are widely used for biodiesel, soap production, and as a condiment in culinary applications. Additionally, harvested Niger plants serve as green manure, improving soil health. However, Niger cultivation in India has significantly declined over the past decade due to low yields, a poor seed set, self-incompatibility, a low harvest index, and seed shattering. A lack of genetic diversity further restricts breeders from developing high-yielding cultivars. The discovery of hereditary male sterility in India has facilitated heterosis utilization and laid the foundation for breeding improved varieties. This review explores the use of traditional breeding methods and genetic tools to enhance Niger, emphasizing plant tissue culture, molecular markers, and the identification of promising gene targets to produce desirable traits. Advanced technologies like CRISPR/Cas, including base and prime editing, are promising to revolutionize Niger breeding and functional genomics research. The insights in this paper stress the urgent need to genetically improve Niger and other underutilized oil crops to meet the increasing global demand for sustainable and diverse oil crops. Such interventions could transform Niger cultivation, ensuring its role in global agriculture.

1. Introduction

Niger [Guizotia abyssinica (L. f.) Cass.] is a diploid species with 2n = 30 and a genome size of about 3.8 pg [1]. Niger is an important minor oilseed crop in India, Kenya, Uganda, Sudan, Malawi, and other nations on the African continent and Indian subcontinent. It is a dicotyledonous herb, moderately to well branched, and grows up to 2 m tall. The seed contains about 40% oil with a fatty acid composition of 75–80% linoleic acid, 7–8% palmitic and stearic acids, and 5–8% oleic acid [2]. The Indian types contain 25% oleic and 55% linoleic acids [3]. Niger oil is also used as a substitute for olive oil. Niger oil has good keeping quality, has <70% unsaturated fatty acids, and is free from toxins and therefore considered good for health [2]. Niger has been recognized as a ‘neglected and underutilized’ crop [4], and thus, it has received little attention from researchers. Niger has the potential to contribute towards achieving self-sufficiency in vegetable oil production in India. Niger oil can be utilized as a paint, lubricant, or animal feed, and it is incorporated in the field as a fertilizer. Low-yielding cultivars, self-incompatibility, lodging and shattering problems, susceptibility to pests, etc., are the major constraints to developing high-yielding cultivars through traditional plant breeding programs [4]. Biotech crops, which include transgenic, genetically modified crops or non-transgenic crops made through DNA-free gene editing, help make or modify a product for a practical purpose. This crop can help solve crop production problems that traditional technologies have not been able to, and it can be used to improve yield and production through biotechnology, genomics, and marker-assisted breeding methods [5].

This review covers the application of conventional breeding, genomics, and biotechnological tools to the underutilized crop Niger for the development of desired trait-specific varieties. CRISPR/Cas genome editing tools have been employed in some oilseed crops to improve their traits [6]. It is also emphasized in this review how important genomic methods are for finding, identifying, and using the desired traits from wild crop relatives (Figure 1).

2. Production Volume of Niger Seed Across India

India ranks first in the production and export of Niger seed [7]. It is cultivated in the Andhra Pradesh, Madhya Pradesh, Orissa, Maharashtra, Bihar, Karnataka, Nagar Haveli, and West Bengal states of India, of which Madhya Pradesh is the largest producer. The graph in Figure 2 shows the highest production in the financial year of 2013 (101,000 MT) and the lowest production in the year of 2024 at 27,000 MT. Starting from the year of 2013, the production of Niger decreased, but in 2017, production slightly increased and declined again. Niger has a high oil content of around 40%, with a valuable fatty acid composition. However, its high unsaturated fatty acid content leads to poor keeping quality, which limits its wider use. Factors such as a lack of government interest in tribal cuisine, limited genetic variation, susceptibility to diseases, a lack of diverse varieties, shattering, self-incompatibility, less or low responsiveness to management inputs [8], and little research attention have mainly contributed to a decline in production, and Niger has mainly remained as an underutilized oilseed crop [9]. There is a need to increase the production of Niger.

In Africa, Niger seed is primarily grown in Ethiopia, with smaller-scale cultivation in Sudan, Uganda, Tanzania, Malawi, and Zimbabwe [10]. Niger seed (Guizotia abyssinica) is a key source of edible oil in Ethiopia, primarily cultivated by small farmers. It contributes about 30% of the country’s oilseed production, with 26% being reserved for home use. While grown across Ethiopia, over 90% is produced in the highlands of Amhara and Oromia. Key production areas include Woollega and Shewa in Oromia and Gojjam and Gondar in Amhara. The main cultivated varieties are Abat, Mesno, and Bunegne [10].

Production Volume of Niger Seed in Ethiopia

Niger seed production has a long history in Ethiopia and remains a significant crop, accounting for 50–60% of the country’s oil crop output. According to the Central Statistical Agency (CSA) of Ethiopia, over the last 18 years, its production has doubled, rising from 1.04 million quintals to approximately 3 million quintals. This improvement is likely due to the adoption of improved varieties and enhanced agronomic practices, such as fertilizer application, timely planting, site selection, and effective field management [11]. This suggests a potential to boost production and productivity by developing suitable varieties and improving agronomic practices through breeding and biotechnological tools.

3. Limitations of the Crop

Niger has an indeterminate growth habit and an extremely low harvest index [12]. Lodging tendency and shattering of the seed directly affect the seed yield. Due to its self-incompatible nature, it is difficult to develop an inbred line, and conventional breeding works in Niger are very difficult to conduct [13]. However, using techniques for population improvement should lead to steady genetic gain, and the types that are self-compatible that are found in Ethiopia can be used to make high-yielding synthetic populations or even hybrids. Sib mating plays an important role in developing inbred lines; hence, it can help develop selfed seeds (self-compatible types). Insects and pests like Niger flies attack Niger flowers, which feed on flower heads and interfere with seed-setting and pollination, causing a direct reduction in seed set, have been observed [14,15,16]. The insects black pollen beetles feed on flower heads, thereby reducing seed set [16]. Compared to other oilseed crops, Niger is less prone to disease occurrence. Diseases such as Niger stem and leaf blight can be a problem in modernly released cultivars. Niger seed has a high fiber content, and crushers and processors have reported that it has a lower oil recovery rate than other oilseeds. A report showed that animals that are fed Niger meal gain less weight than those fed meals made with other oilseeds, although there is no known toxic or anti-nutritional factor present in Niger [7].

4. Conservation of Niger Germplasm

The National Bureau of Plant Genetic Resources (NBPGR), India, has achieved the ex situ long-term conservation of germplasms in India. The base collection is preserved for long-term storage at −20 °C, whereas medium-term storage is carried out at 4 °C. It is crucial to determine the percentage of genetic diversity represented within these conserved collections, identify what remains unconserved, and develop strategies for utilizing the available resources. The NBPGR holds 1807 Niger germplasm accessions [17]. Threatened resources, particularly wild species, should be prioritized. Due to its self-incompatibility, Niger is a cross-pollinated crop. To keep its germplasm safe, it is kept in several places by siblings (grouping plants together) to stop accessions from crossing with each other (Figure 3).

In India, genetic diversity has been harnessed by the direct selection of locally adapted material. So far, improved plant breeding technologies have been used the least in Niger. The genetic basis of cultivars is limited. Therefore, germplasm utilization must be enhanced for the genetic enhancement of Niger. Crop wild relatives or germplasms serve as resources for enhancing genetic performance in crop plants by offering a comprehensive reference of genetic diversity to enhance certain traits. Therefore, wild species can be used in crop trait improvement [18]. The use of breeding and biotechnology has had a large effect on expanding the genetic base of Niger germplasms for a wide range of agronomic traits and has made it possible to create new cultivars with better quality and quantity traits [19].

5. Breeding and Crop Improvement

The heterogeneous seed population of Niger in India offers significant potential for yield improvement through various breeding programs. Key objectives include increasing the yield and oil content while reducing shattering. Lowering the plant height improves the harvest index by reducing the leaf number and enhancing fertilizer efficiency, thus boosting seed yields. Due to its self-incompatibility, Niger is suitable for hybrid variety development, like the sunflower. The discovery of genetic male sterility in India [20] has enabled the exploitation of heterosis in Niger.

Mutation breeding using chemical and physical mutagenesis has created genetic variability for developing trait-specific varieties [21,22]. Mutants with traits like high oleic acid and oil contents have been isolated, with four superior lines (OY-155, OY-325, HO-308, and OO-309) being developed for agricultural use. Increased oleic acid levels resulted from induced mutations affecting oleic acid desaturation, reducing polyunsaturated fatty acid contents [23]. Wild species of Guizotia (G. scabra subsp. Schimperi) provide disease resistance genes for introgression into cultivated species [2]. Mutation and transgenic breeding form key pillars of modern Niger breeding programs [24,25]. Misal et al. 2021 [25] studied and inferred that backcross breeding has the potential to develop high-yielding cultivars of Niger, highlighting its importance for sustainable food security. In 2020, Amsalu [26] revealed that the presence of a considerable magnitude of variability among the different genotypes of Niger for all of the traits studied can be incorporated in the breeding program to develop a high oil yield, a high seed yield, and early-maturing cultivars. Similarly, the genetic divergence among tested genotypes suggests the potential to identify diverse materials with desirable agronomic traits, enabling the development of superior Niger seed cultivars through the hybridization and selection of accessions from different clusters [8].

The priority of the Niger crop improvement program is to increase the seed oil content and overcome the problems of pod shattering and self-incompatibility. The germplasm with a diverse oil content is available in Ethiopia and India, and it can be incorporated into the breeding program to significantly increase the oil content. Pollination behavior favors the genetic improvement program in the Niger crop. The recurrent selection method is used as a standard breeding procedure for the development of open-pollinated varieties because of their self-incompatibility. Thus, Niger is a candidate for hybrid development. The identification of genetic male sterility in India and recently in Ethiopia has paved the way for the exploitation of heterosis in Niger.

6. Biotechnological Interventions in Niger’s Improvement

Using biotechnology to improve crops is very important because it helps with gene pyramiding, genetic diversity assessment, genomics-assisted speed breeding, and genetic modifications that increase the nutritional value, oil content, and tolerance to biotic and abiotic stresses. The challenge of self-incompatibility in Niger complicates the development and maintenance of inbred lines, as noted by Getinet and Teklewold [2]. This biological barrier prevents self-fertilization, which is critical for achieving genetic uniformity in breeding programs. However, in vitro technologies offer a promising alternative for genetic improvement. These methods include techniques such as in-vitro culture, somatic embryogenesis, and direct regeneration, which allow for the propagation and manipulation of plants under controlled conditions [2]. A reliable plant regeneration system is pivotal to the success of in vitro approaches. It ensures the consistent and efficient development of plants from explants, somatic cells, or tissues, thereby facilitating the genetic upgrading of Niger by bypassing the limitations imposed by its self-incompatibility. In vitro technology, such as plant tissue culture, serves a major role in the plant regeneration system [18]. Somatic embryogenesis is the successful method utilized for plant regeneration in Niger plants from suspension cultures [27]. Agrobacterium-mediated genetic transformation has a significant role in the transfer of specific desired genes in crops. Biotechnological interventions for studying and improving Niger are elaborated in detail below.

6.1. Plant Tissue Culture

Plant tissue culture in Niger offers a promising avenue for agricultural advancement. This technique enables the rapid propagation of elite varieties, disease-free plant production, and genetic improvement of crops’ crucial traits [28,29]. Niger has been extensively studied in cell and tissue cultures, with regeneration reported from various explants, including leaves [30,31,32], anthers [33,34,35], cotyledons, and hypocotyls [36,37,38]. Tiwari et al. [5] highlighted the utility of anther culture in developing homozygous lines, which can be incorporated into breeding programs for hybridization. It was found that somatic embryogenesis (Figure 4) through suspension cultures has great potential. It can help carry out in-vitro variant selection and crop improvement for crops that are tolerant to biotic and abiotic stresses [27].

It was reported that through anther culture, haploid production and the development of large flower heads, dwarf, and self-compatible plants were developed, which are useful for Niger improvement, particularly to overcome the problems of self-incompatibility and low yield [34]. Jadimath et al. [32] effectively maintained male sterile plants through the tissue culture method and used explants as leaves of a male sterile plant that were developed through gamma irradiation. Anther culture is used for the development of di-haploid plants and self-compatible lines such as dwarf and single-headed double-haploid plants. Anther culture-derived di-haploid plants can be used to develop homozygous inbred lines within a short time [19]. Niger has been the focus of various cell and tissue culture studies, emphasizing its potential for crop improvement and conservation. The regeneration of plantlets through tissue culture has been successfully reported using specific explants like cotyledons and hypocotyls [33,36,37]. Advancing tissue culture techniques can address some of the crop’s inherent challenges, such as low yield, disease susceptibility, and adaptability issues, thereby unlocking its full potential for agricultural and industrial applications [39]. The use of anther culture and tissue culture techniques in Niger is promising for several future advancements. These methods can enhance breeding efficiency by rapidly developing homozygous inbred lines, improving the yield and self-compatibility traits.

In vitro techniques are important tools for crop improvement and have led to the induction of somaclonal variations [40]. Several methods, including karyotyping and isoenzyme profiling, are employed to assess the genetic fidelity of in vitro-derived clones. However, these approaches have inherent limitations. Karyotyping does not reveal the chromosomal rearrangement and alteration in the genes, whereas isoenzyme profiling is subjected to ontogenic variations [41]. Therefore, it is of utmost importance to deploy molecular markers to detect the variations in the genes or genetic relationships among individuals between and within species. Integration with genetic transformation methods may enable the introduction of traits such as disease resistance and environmental stress tolerance. Overall, these biotechnological tools can drive significant improvements in the productivity and sustainability of Niger cultivation.

6.2. Molecular Marker Technology

Molecular markers provide immense support to plant breeding and significantly accelerate crop improvement efforts [42,43]. These markers have a crucial role in the characterization and evaluation of available germplasms through DNA fingerprinting, the estimation of genetic variability, and genetic diversity [43]. The potential for crop improvement through breeding depends on the magnitude of genetic diversity and genetic variation. It is of utmost importance to characterize and evaluate the inherent value of the conserved germplasm. Accessible collections of varied wild and cultivated germplasm accessions have greatly aided in crop improvement [5]. Molecular markers have immense potential for the exploration of genetic diversity and variation and for interpretation in polymorphic form [44,45,46]. RAPD markers have predominantly been used for the study of phylogenetic relationships in Niger and its wild relatives. Geleta et al. [47] investigated the genetic diversity and variation of 70 populations of Ethiopian Niger through RAPD markers and found 97% polymorphism. This study revealed that the genetically distant populations that were confined to different clusters are unique and can be incorporated into the marker-assisted breeding program for crop improvement. Nagella et al. [48] studied the genetic diversity of a few Indian Niger germplasm accessions using RAPD markers. It was the first attempt to use molecular markers to quantify genetic diversity among cultivars of Indian Niger. Dempewolf et al. [49] developed a library of EST and designed 43 SSRs using EST sequences and the sequence from the chloroplast genome. These microsatellite markers were explored to assess the genetic diversity and genetic variation among the diverse genotypes, which led to a better understanding of phylogeographic patterns. Sanger sequencing was used to yield a full chloroplast genome and was submitted to Genebank (GeneBank id EU549769⁄Refseq NC 010601) after annotation. In the same way, Tsehay et al. [1] identified SNP markers through the transcriptome sequencing of two genotypes, leading to KASP assays for 554 SNPs for genotyping in Niger. There are currently no global reports of trait mapping or marker-assisted breeding in Niger, which can target traits such as abiotic stress tolerance, disease resistance, yield, and quality [50].

Future research should focus on creating high-density genetic maps that integrate various marker types (SNPs, SSRs, etc.) for precise QTL mapping of key agronomic traits, such as oil yield, disease resistance, and stress tolerance. This integrated map, combined with phenotypic data from diverse germplasms, will support robust marker-assisted breeding strategies to accelerate the improvement of Niger. Additionally, the complete chloroplast genome sequence opens opportunities for chloroplast transformation in genetic engineering. Research should aim to combine these molecular resources with advanced breeding techniques to efficiently introduce desirable traits into Niger varieties, enhancing productivity and addressing food security challenges.

6.3. Genetic Transformation

Genetic transformation provides a means to address many challenges and improve key traits in crops. But the development of efficient transformation protocols remains crucial for the successful implementation of genetic engineering strategies in crops, including Niger. To improve the key traits of Niger and its oil, it is essential to establish efficient transformation protocols for genetic improvement. To overcome constraints such as self-incompatibility, shattering, and susceptibility to diseases, the genetic improvement of Niger is essential [4,39]. The success of such genetic improvements depends on the establishment of a robust and efficient transformation protocol [39]. Genetic transformation is a technique where a gene of interest is transferred or inserted into a desired genotype.

Only one report is available so far on the Agrobacterium-mediated genetic transformation of Niger. Murthy et al. [39] reported the protocol for the Agrobacterium-mediated genetic transformation of Niger using hypocotyl and cotyledon explants of the UNS-4 variety. Seven-day-old seedlings were used as explants for transformation. The explants were cultured on MS medium with varying concentrations of 6-benzylaminopurine (BA) for shoot regeneration. Agrobacterium tumefaciens strain EHA101, with the binary vector pIG121Hm, was used for transformation. The T-DNA region contained nptII and hpt genes as selectable markers and GUS as a reporter gene, driven by the CaMV 35S promoter [39]. Co-cultivation occurred for 3 days at 22 °C. Following co-cultivation, the explants were transferred to selection and regeneration media. After successful regeneration, the plants were acclimatized in a greenhouse [39]. The Agrobacterium-mediated transformation of Niger using seedling explants is depicted in Figure 5.

In this study, the transformation frequency of cotyledon was 15%, whereas the transformation frequency of hypocotyl was only 3% [39]. No other report on Niger’s transformation is currently available. Future research should focus on developing high-throughput, efficient, and cost-effective transformation systems. This necessitates optimizing existing protocols, exploring novel gene-editing technologies like CRISPR/Cas9, and investigating alternative explant sources. To improve Niger genetic transformation efficiency, a multi-faceted approach is necessary. Expanding the range of explants tested beyond those used by Murthy et al. [39] is crucial. Different tissues may have varying responses to transformation, necessitating the optimization of tissue culture conditions for regeneration. This involves refining media composition, hormone concentrations, and culture parameters to improve shoot and root development from transformed explants. A comparative analysis of Agrobacterium strains is important due to their differing virulence and transformation efficiency. Exploring diverse plasmids with various selectable markers and replication origins will enhance transformation success. Additionally, testing different promoters for transgene expression is crucial for consistency. A systematic investigation of these factors will help identify optimal conditions for efficient and reliable Niger transformation.

7. Genome Editing Overview and Its Applications in Niger Crops

CRISPR-based genome editing methods, including CRISPR/Cas9, CRISPR/Cpf1, base editing, and prime editing, have shown promise in improving crop genetics and elite characteristics [51]. CRISPR-Cas proteins, including fCas9, Cas12, Cas13a, and Cas13b, offer new prospects for the enhancement of crop research [52,53,54,55,56,57]. CRISPR/Cas systems modify eukaryotic DNA by creating a guide RNA (gRNA) that matches the target sequence. This gRNA has a protospacer motif of around 20 base pairs, bordered by site-specific homology sequences. In the CRISPR-Cas9 system, Cas9 binds to a protospacer motif in gRNA and forms a complex with the target site. Cas9 interacts with the protospacer adjacent motif (PAM). The PAM catalyzes a double-strand break (DSB) in the host DNA, with three base pairs upstream [58]. The host cell’s DNA repair machinery can mend the cut by either homology-directed repair (HDR) or non-homologous end joining (NHEJ) [59]. The NHEJ repair may lead to small deletions, and insertion can cause gene function knockouts and knockdowns, whereas HDR can result in DNA sequence modifications, including gene knock-ins or gene replacements (Figure 6).

Other CRISPR-Cas-based systems, such as base editing and prime editing, may modify DNA sequences through the chromatin structure or base editing, eliminating the requirement for cuts to DNA strands [57]. Because of its adaptability, CRISPR-Cas9 technology has emerged as a valuable tool in genome editing and can play an important role in studying gene functions and improving Niger and other oilseed crops (Figure 7). There is a need for understanding or deciphering the genes involved in pod dehiscence as it is causing major loss in oilseed crops [60]. Pod dehiscence significantly reduces yield in oilseed crops, including soybean, sesame, and oilseed rape, both before and after harvest. Oilseed crop breeding aims to minimize shattering loss. Mutant research has identified the genes responsible for shattering resistance. Minimizing pod shattering through biotechnology is a top objective. The identification of genes involved in pod dehiscence is necessary to develop the plant’s tolerance or resistance to pod shattering. For instance, in Brassica napus, a comprehensive approach led to the discovery of BnTCP8.C09 as the gene accountable for pod shattering [61]. The removal of the pod dehiscence 1 (PDH 1) gene led to decreased pod shattering in soybean [62]. Self-incompatibility is another constraint that limits the production and productivity of oilseed crops. Pollination and fertilization result in the production of seeds, which is the economically valuable aspect of oilseeds. The presence of self-incompatibility impedes the development of inbred lines, and overcoming self-incompatibility is a key breeding goal in oilseed crops like Niger. Researchers have successfully achieved self-incompatibility by modifying important genes such as S-receptor kinase and M-locus protein kinase in oilseed rape [63].

Harnessing genome editing technology led to a significant development in oilseed crops with improved yields and productivity, thereby contributing to food security and sustainable agriculture. The combination of these findings strongly suggests that genome editing techniques have created promising opportunities for enhancing oilseed crops, and we anticipate that this research will have significant benefits in the future [64]. However, there are also several challenges in the application of CRISPR-based genome editing technology in oil crops. Genome complexity and recalcitrance to transformation, along with a polyploidy nature, led to the great challenge of efficiently obtaining CRISPR-based knockout lines in some oil crops, especially sesame, sunflower, and groundnut. Thus, novel genetic element delivery systems for these transformation-recalcitrant oil crops must be developed in the future [64].

Gene targeting technology in plants predominantly utilizes homology-directed repair (HDR) to achieve precise genome editing. This involves the introduction of specific insertions, sequence replacements, or nucleotide substitutions. However, the low efficiency of HDR has constrained its broader application in plant genetics [65,66]. To address these limitations, alternative genome editing technologies such as deaminase-mediated base editing and reverse transcriptase-mediated prime editing have emerged as promising methods. Unlike traditional HDR techniques, these CRISPR–Cas-based approaches do not create double-strand breaks (DSBs) and do not require the incorporation of donor DNA. This characteristic not only enhances their efficiency compared to HDR but also allows for precise editing without the complications associated with DSB formation. As a result, these innovative technologies are becoming increasingly valuable for improving genetic engineering in plants, providing more reliable and efficient means of achieving desired genetic modifications [51]. The below sections discuss the cytosine base editor (CBE), adenine base editor (ABE), and prime editing tools.

7.1. Cytosine Base Editing (CBE)

Cytosine base editing (CBE) is an advanced genome editing technique that leverages a modified form of the CRISPR-Cas9 system to achieve precise edits in plant genomes. Specifically, CBE utilizes a Cas9 nickase (nCas9) with a D10A mutation, which inactivates one of the nuclease domains, RuvC, allowing for single-strand breaks (or nicks) rather than double-strand breaks (DSBs). This modification minimizes the potential for unwanted genomic damage that can result from traditional Cas9 nuclease activity [67].

In CBE, the nCas9 is fused with two key proteins: a cytidine deaminase, which is responsible for converting cytidine (C) into uridine (U), and an uracil DNA glycosylase (UDG) inhibitor (UGI), which prevents the repair of the uracil that has been generated. As a result, when a C:G base pair is targeted by a specific single guide RNA (sgRNA), the deaminase effectively changes the cytosine to uracil, and subsequently, during DNA replication or repair processes, the uracil can be replaced with thymine (T). This enables the conversion of C:G base pairs into T:A pairs, achieving precise base transitions without the need for donor DNA and circumventing the limitations associated with homologous recombination [51].

The efficiency and precision of CBE make it a powerful tool in plant genome editing, expanding the possibilities for creating genetically modified plants with desired traits. CBE can be used to introduce beneficial mutations in Niger genes related to oil content, disease resistance, and abiotic stress tolerance, improving its overall productivity and sustainability (Figure 7). This technology can help overcome limitations in traditional breeding by enabling precise modifications in specific genes without the need for extensive backcrossing.

7.2. Adenosine Base Editing (ABE)

Adenosine base editors (ABEs) represent a significant advancement in the precision of genome editing, particularly for achieving A:T to G:C substitutions in plant genomes. This innovation utilizes an engineered version of adenosine deaminase, specifically derived from the Escherichia coli tRNA-specific adenosine deaminase (ecTadA), which has been optimized to function effectively in a CRISPR/Cas9 [68]. In the ABE framework, the adenosine deaminase is fused with a catalytically inactive variant of Cas9 (nCas9, with the D10A mutation), which allows for targeted editing without creating double-strand breaks (DSBs) in the DNA. When the ABE complex binds to a particular genomic target, the adenosine deaminase component facilitates the conversion of adenosines into inosines. During subsequent DNA repair and replication processes, inosines are interpreted as guanosines by DNA polymerases. This leads to the desired A:T to G:C transition, allowing for precise nucleotide substitution without the need for donor DNA or additional repair mechanisms [51]. The development of ABEs has addressed some of the limitations associated with homology-directed repair (HDR), as they can achieve high levels of editing efficiency with lower potential for off-target effects.

ABE offers a valuable tool for enhancing Niger’s agronomic traits by enabling precise A-to-G base changes in genes associated with yield, quality, and stress tolerance. This technology can be particularly useful for addressing mutations that cause undesirable traits in Niger, leading to improved crop performance (Figure 7).

7.3. Prime Editing

Prime editing is an advanced genome editing technique that integrates CRISPR technology with reverse transcription for accurate genetic modifications. It uses a modified Cas9 protein, nCas9 (H840A), which creates a single-strand break in DNA without cutting both strands, enabling precise editing. The prime editing guide RNA (pegRNA) directs nCas9 to the target site and includes a reverse transcriptase template that holds the desired mutations and a primer-binding site that pairs with the single-stranded DNA to initiate reverse transcription. This process synthesizes DNA using the RNA template, incorporating the genetic edits directly into the genome [51,69]. Thus, prime editing provides a versatile and accurate method for making targeted genetic changes with fewer off-target effects. Prime editing provides a versatile platform for diverse genetic modifications in Niger, including insertions, deletions, and precise nucleotide substitutions, enabling the creation of novel and improved varieties. This technology offers the potential to address complex genetic traits and introduce more sophisticated genetic modifications than previously possible in Niger (Figure 7).

Moving forward, the future direction of genome editing in oilseed crops should focus on refining delivery systems and methodologies to overcome the challenges of transformation recalcitrance and genome complexity. Advances in nanoparticle-based delivery methods and viral vectors could offer more efficient means of introducing CRISPR components into target cells, thereby enhancing transformation rates. Integrating multi-omics approaches, such as genomic, transcriptomic, and proteomic analyses, will help in understanding the underlying mechanisms of gene regulation and expression patterns in these crops. By addressing these challenges and improving delivery mechanisms, we can unlock the full potential of CRISPR technology, paving the way for the development of oilseed varieties with enhanced traits, ultimately contributing to global food security and sustainable agricultural practices.

8. Future Strategies

Ethiopian and Indian Niger populations are genetically diverse, offering the potential for breeding programs to improve production and productivity. Key breeding objectives include improving the seed yield, oil content, self-incompatibility, disease resistance, and shattering. To address self-incompatibility, breeders in Ethiopia and India use mass selection and sibbing while screening for lines with minimal inbreeding depression. Wild Guizotia species can provide disease resistance genes for introgression into cultivated Niger [4]. The exploitation of heterosis will soon be possible through the development of male sterile lines and inbreds. This has opened a new dimension in heterosis breeding in Niger improvement [9]. The major constraints in Niger are self-incompatibility, shattering, the development of high-yielding cultivars, cultivars with a high oil content, and disease resistance. These can be overcome through tissue culture approaches, such as somatic embryogenesis, and by using marker-assisted breeding and genome editing technologies. CRISPR-based genome editing can accelerate the domestication of wild oil crops and improve cultivated ones to meet the growing demand. However, its application in oil crops faces challenges, including difficulties in creating CRISPR-based knockout lines in crops like sesame, sunflower, and peanut due to complex genomes and low transformation frequency. Producing thoroughly mutated lines in polyploid crops like rapeseed is also a significant challenge [64]. Base editing and prime editing offer powerful tools for precise genetic modification in Niger [64]. CBEs and ABEs can be used to correct specific point mutations associated with undesirable traits, such as disease susceptibility or reduced yield. Precise editing of the BnALS1 gene at position P197 in oilseed rape was achieved using a CBE system, which efficiently introduced C to T conversion in the sgRNA target (Figure 7). An editing efficiency of 1.8% was recorded, resulting in P197S substitution in BnALS1, which created a novel herbicide-resistant mutant in oilseed rape, facilitating weed management [70]. Such studies can be undertaken in Niger with powerful CRISPR/Cas tools to improve the traits and oil contents in the future.

Prime editing’s precision enables the introduction of beneficial alleles or the modification of regulatory sequences to enhance gene expression, improving key agronomic traits (Figure 7). These technologies can accelerate the development of resilient, high-yielding Niger varieties. Applications include boosting the yield, enhancing disease and herbicide resistance, inducing haploids, generating male sterile lines, fixing hybrid vigor, manipulating self-incompatibility, and facilitating de novo domestication. This technology is set to transform oil crop cultivation and breeding practices [64]. CRISPR technology holds immense potential to transform Niger. By precisely editing crop genomes, it can enhance drought and disease resistance, boost the oil content, and improve herbicide tolerance, leading to more resilient and productive Niger crops.

9. Conclusions

Niger is an oilseed crop in India, accounting for more than 50% of the world’s Niger area and production. Niger is treated as a neglected underutilized crop despite being nutritionally rich, medicinally important, and economically important for the livelihood of small and marginal farmers in arid and semi-arid areas of the country. Niger is an important minor oilseed crop that can be used to develop better oil quality and high seed yield in Niger cultivars. The molecular breeding strategy helps to overcome constraints such as low-yielding cultivars, self-incompatibility, lodging and shattering problems, susceptibility to pests, etc. For a long time, Niger has remained a neglected and underutilized crop, and because of this, it has received little attention from the scientific community. The in-vitro micropropagation and tissue culture method is indeed a cornerstone technology in plant breeding, offering a controlled and efficient method to produce large quantities of genetically uniform plants. However, during this process, somaclonal variation plays an important role in crop improvement. By integrating somaclonal variation into modern breeding pipelines and combining it with genomic tools, breeders can uncover and stabilize new genetic diversity to meet the growing challenges of agriculture. Explorations of germplasms through collections do not merely provide an opportunity to broaden the genetic base of these crops in gene bank collections. The government has initiated several programs to improve yields through better varieties, with consequent benefits to farmers and to the country through an increased supply of seed available for export. A lack of comprehensive research, omics approaches, and studies on genetic transformation and QTL mapping represent major research gap. To bridge these gaps, the Department of Biotechnology, Govt. of India [71], undertook the mission mode program titled ‘Minor Oilseeds of Indian Origin’ to harness their benefits to the communities through the interventions of cutting-edge genomic technologies. Such initiatives foster research on underutilized minor oilseed crops like Niger in the domain of omics and marker-assisted breeding approaches, which have been late entrants into the genomics era. An environment-neutral approach, such as molecular marker techniques, seems to be a better option to include in crop improvement programs for Niger. Efforts are being made to use molecular markers in Niger to study its genetic diversity and abiotic stress responses. The demand for industrial applications and oil consumption is increasing day by day over time. Therefore, Niger’s yield, its quality, its oil content, and its traits related to biotic and abiotic stresses should be improved. However, the crop is facing different production and breeding constraints. The extension of biotechnological tools and the presence of high genetic variation create the opportunity to improve the crop with available resources. Biotechnology tools can be very useful for breeding Niger crops and improving them by adding desirable traits using genomics and in vitro techniques. It is expected that genomics will soon bring rapid progress in Niger research.

Author Contributions

Conceptualization, J.N.P. and S.S.G.; writing—original draft preparation, J.N.P., K.P.I., N.R.T. and M.P.; writing—review and editing, K.P.I., N.R.T. and S.A.C.; visualization, N.R.T. and S.A.C. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

No new data were created or analyzed in this study. Data sharing is not applicable to this article.

Conflicts of Interest

Author Krishnananda Pralhad Ingle was employed by the company Reliance Industries Limited. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Footnotes

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Figures

Figure 1. Breeding and molecular strategies for the development of desired trait-specific cultivars of Niger. The selection, characterization, and screening of genetic resources, such as cultivated and wild species, landraces, and neglected underutilized crops, are carried out through traditional and modern breeding techniques and biotechnological interventions to develop trait-specific resilient cultivars/varieties of Niger. Precision phenotyping can be used to understand the physiology and genetics of the breeding germplasm and then be developed into high-throughput phenotyping assays and potentially molecular markers for rapid screening in Niger. High-throughput genotyping identifies the genetic variations among the superior lines. Genomic selection, an advanced marker-assisted breeding tool in plant breeding methods, is one of several approaches that meet these requirements. It uses genome-wide markers, phenotype information, field trials, and the identification of superior lines. The identified candidate genes and markers related to the desired traits found from high-throughput sequencing and phenotyping data analysis will allow for the recombination of superior plant genotypes introduced in the cropping system to develop desired trait-specific cultivars/varieties of Niger.

Figure 2. Production volume of Niger seed across India from 2013 to 2024 (in 1000 metric tons) (source: https://www.statista.com/statistics/769707/india-niger-seed-production-volume).

Figure 3. A drone shot of a seed multiplication/regeneration block for Niger breeding at the Indian Council of Agricultural Research-National Bureau of Plant Genetic Resources (ICAR-NBPGR) Campus, Akola, India, dated 15 December 2020. These are the 1700 different Niger accessions along with the checks used for seed multiplication. From the left border, 2 rows are checks JNS 28 and JNS 30; the middle 9 rows are 1700 accessions for seed multiplication; and from the right border, 2 rows are checks JNS 9 and IGPN 2004-1.

Figure 4. Deployment of somatic embryogenesis in underutilized Niger crops. (a) Embryogenic nodular calli induced from immature embryo explants. (b) Embryogenic cells of suspension culture that underwent division and formed cell clusters. (c) Germination of somatic embryos. (d) Shoot and root development. (e) Plant regenerated from somatic embryo and transplanted in greenhouse for acclimatization. (f) Matured Niger plant with flower setting.

Figure 5. Protocol for Agrobacterium-mediated transformation of Niger. Step (1), optimization of media for shoot regeneration; step (2), Agrobacterium tumefaciens strain (e.g., EHA101) preparation for genetic transformation; Step (3), co-cultivation of explants with Agrobacterium; step (4), transfer on selection medium (MS medium containing 1 mg/L BA, 50 mg/L kanamycin, and 50 mg/L hygromycin); step (5), separation of resistant and elongated roots (3–4 cm long) and transfer to rooting medium; step (6), hardening and transfer to greenhouse; step (7), plant maturation; step (8), molecular and phenotypic analysis of transgenic plants (created in https://BioRender.com).

Figure 6. CRISPR/Cas9 for DSBs and repair either by Non-Homologous End Joining or Homology Directed repair. Cas9 is a protein that acts as a molecular scissor that binds to guide RNA. The guide RNA has RNA bases that are complementary to those of the target DNA sequence in the genome. The protospacer adjacent motif (or PAM for short) is a short DNA sequence (usually 2–6 base pairs in length) that follows the DNA region targeted for cleavage by Cas9. The PAM is required for a Cas nuclease to cut. Cas9 cuts both strands of DNA at a specific site in the genome. This cut creates a double-strand break (DSB), which the cell then attempts to repair. Depending on the repair mechanism used by the cell, new sequences can be introduced (via HDR, homology-directed repair) or mutations can be introduced (via NHEJ, non-homologous end joining). NHEJ introduces semi-random insertion–deletion mutations (indels), whereas HDR (Homology Directed Repair) achieves the precise insertion, deletion, or substitution of nucleotides using donor templates.

Figure 7. Application of CRISPR/Cas-based tools in Niger for crop improvement. The primary biotic and abiotic factors that hinder the growth and yield of Niger crops include pest infestations, fungal diseases (such as blast), a low oil yield, seed shattering, a high fiber content with a low oil content, and self-incompatibility. The second panel outlines the comprehensive strategy employed to validate the target gene’s role in Niger. The approach integrates genome-wide association studies (GWAS), transcriptome analysis, quantitative trait locus (QTL) mapping, and quantitative real-time PCR (qRT-PCR) to identify and characterize the gene’s function. The third panel depicts the application of advanced genome editing techniques, specifically CRISPR-Cas-based approaches, to modify the target gene in Niger. The three primary strategies utilized are adenosine base editing (ABE) and cytosine base editing (CBE), which precisely convert specific DNA base pairs without introducing double-strand breaks. Prime editing allows for a wide range of genetic modifications, including insertions, deletions, and base substitutions. The last panel illustrates the workflow for generating and characterizing genome-edited Niger plants. The process involves genome editing, genotyping to identify desired mutations, phenotyping to assess the impact of the edits on plant traits, field trials to evaluate performance under real-world conditions, and, ultimately, the release of improved Niger cultivars (the figure was created in https://BioRender.com).

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