Progress in genetic engineering and genome editing of peanuts: revealing the future of crop improvement

Abstract

Peanut (Arachis hypogaea L.), also known as groundnut, is cultivated globally and is a widely consumed oilseed crop. Its nutritional composition and abundance in lipids, proteins, vitamins, and essential mineral elements position it as a nutritious food in various forms across the globe, ranging from nuts and confections to peanut butter. Cultivating peanuts provides significant challenges due to abiotic and biotic stress factors and health concerns linked to their consumption, including aflatoxins and allergens. These factors pose risks not only to human health but also to the long-term sustainability of peanut production. Conventional methods, such as traditional and mutation breeding, are time-consuming and do not provide desired genetic variations for peanut improvement. Fortunately, recent advancements in next-generation sequencing and genome editing technologies, coupled with the availability of the complete genome sequence of peanuts, offer promising opportunities to discover novel traits and enhance peanut productivity through innovative biotechnological approaches. In addition, these advancements create opportunities for developing peanut varieties with improved traits, such as increased resistance to pests and diseases, enhanced nutritional content, reduced levels of toxins, anti-nutritional factors and allergens, and increased overall productivity. To achieve these goals, it is crucial to focus on optimizing peanut transformation techniques, genome editing methodologies, stress tolerance mechanisms, functional validation of key genes, and exploring potential applications for peanut improvement. This review aims to illuminate the progress in peanut genetic engineering and genome editing. By closely examining these advancements, we can better understand the developments achieved in these areas.

Introduction

Peanut (Arachis hypogaea L.) belongs to the legume family (Fabaceae). A. hypogaea (AABB) is an allotetraploid (2N = 4x = 40) crop that emerged from hybridization between A. duranensis (AA genome) and A. ipaensis (BB) (Kochert et al. 1991; Bertioli et al. 2016). The genome size of peanuts is approximately 2.54 Gb, containing 83,709 protein-coding genes (Chen et al. 2019; Zhuang et al. 2019). As a leguminous plant, peanuts can fix atmospheric nitrogen in their root nodules through symbiotic interactions with microorganisms. In contrast to other leguminous crops that typically produce pods above ground, peanut uniquely develop their pods underground (geocarpy) (Ozias‐Akins and Breiteneder 2019). Peanuts are cultivated on approximately 30 million hectares of land worldwide for food and feed purposes (FAO 2023). In 2022, the global peanut production, excluding shelled peanuts, reached an impressive 54.23 million tons. China holds the top position, contributing 34.88% of the total global peanut production, followed by India (18.68%) and the United States (4.66%) (FAO 2023). In the United States, 2.52 million tons (4.66%) of peanuts were produced on 0.56 million hectares of cultivated land in 2022. With an average yield of 4,500 kg per hectare, the USA is considered one of the leading peanut producers (FAO 2023). Within the United States, the cultivation of peanuts primarily revolves around four main commercial types: Runner (80%), Virginia (15%), Spanish (4%), and Valencia (1%) (Bhogireddy et al. 2020).

Peanuts are widely grown for oil production and consumption. On average, a total of 100 g of peanut seeds serves 567 Cal of energy, 7% of water, 49.2 g total fats (50% monounsaturated, MUFAs; 33% Polyunsaturated, PUFAs; and 14% saturated), 16.1 g carbohydrates, 25.8 g proteins, 8.5 g fiber, and 4.7 g of sugar (Arnarson 2023). In addition, it is a rich source of minerals, essential vitamins, and antioxidants. Peanuts are eaten raw, roasted, boiled, in powder form, peanut butter, peanut oil, and other forms (Settaluri et al. 2012; Arya et al. 2016). Peanuts contain phytonutrients that enhance general health and wellness, such as phenolics, resveratrol, stilbenes, phytosterols, oleic and linoleic acid, folic acid, and iso-flavonoids (Toomer 2018; Zhuang et al. 2019). While peanuts are valued for their nutritional benefits, they can pose significant health risks, primarily through aflatoxin contamination (Meneely et al. 2023). Additionally, peanut allergies are a significant concern worldwide, with more than 1% of the global population (Pandey et al. 2019) and over one million Americans estimated to be allergic to peanuts (Sampson 2004; Chen et al. 2016). Currently, 18 peanut allergens (Ara h 1-Ara h 18) have been identified and designated by the esteemed WHO/IUIS Allergen Nomenclature Subcommittee (WHO/IUIS, 2023). Besides aflatoxins and allergens, peanut cultivation and production face many challenges due to abiotic and biotic stressors.

Traditional approaches and molecular breeding techniques employed for crop improvement have historically been time-consuming, allowing only limited exploitation of natural variations (Khush 2013; Ahmar et al. 2020). To overcome these limitations, mutation breeding has been applied; this technique allows for the creation of previously undiscovered genetic variations and provides the basis for identifying novel traits through forward or reverse genetics. However, it is important to note that these mutations occur randomly within the genome and may inadvertently lead to the loss of crucial gene functions (Lo et al. 2016; Jung et al. 2018). At the same time, genetic engineering offers a means to directly manipulate or introduce agriculturally significant traits into plants, often in a targeted and more time-efficient manner when compared to conventional breeding techniques (Datta 2013; Dong and Ronald 2019). These techniques include DNA transfer mediated by Agrobacterium, a well-established method that facilitates the introduction of foreign genes into plant cells (Fig. 1). In addition to Agrobacterium-mediated transformation, other techniques such as particle bombardment, polyethylene glycol (PEG), and nanoparticles mediated techniques are employed to generate transgenic plants, however these methods are less utilized in peanut transformation due to their limited success. Schematic representation of the Agrobacterium-mediated transformation and regeneration process is illustrated in Fig. 1. This enables to perform gene overexpression, knockdown through RNA interference (RNAi) as well as genome editing using CRISPR technology for precise genetic modifications (Alok et al. 2017; Janga et al. 2017).

Fig. 1.

Schematic representation of the transformation and regeneration process in peanut (Arachis hypogaea) using Agrobacterium-mediated transformation. A Applications: The process enables gene overexpression, RNA interference (RNAi), and CRISPR-Cas9-based genome editing for genetic improvement of peanut traits, such as stress tolerance or disease resistance. B Preparation of Agrobacterium: T-DNA with gene of interest is mobilized into Agrobacterium tumefaciens, which is then cultured at 28 °C and prepared for co-cultivation. C Preparation of Explants: Peanut seeds are soaked in water to facilitate embryo emergence. The embryonic axis and cotyledons are excised from the embryos. D Co-cultivation: The explants are immersed in a liquid co-cultivation medium containing Agrobacterium to facilitate infection, and explants are transferred to solid media for further co-cultivation with Agrobacterium. E Explants are cultured on media designed to induce shoot induction. F The shoots are further grown and elongated in a growth medium. G Elongated shoots are transferred to a medium promoting root development. H Rooted plants are transferred to soil for further growth under controlled conditions

Genetic transformation in peanut

The success of genetic engineering in crops relies on robust regeneration and transformation protocols, often contingent on many factors, including genotype, explant, the strain of Agrobacterium, selection agents, culture media, growth conditions, and phytohormones (Anjanappa and Gruissem 2021). Being a tetraploid and recalcitrant crop, peanuts are difficult to regenerate; therefore, a stable and efficient peanut transformation and regeneration protocol is much needed for crop improvement. To date, various explants are utilized for the optimization of peanut transformation such as leaflets (Halder and Jha 2016), mature cotyledons (Karthik et al. 2018; Huai et al. 2023), immature cotyledons (Deng et al. 2001), de-embryonated cotyledons (Tiwari and Tuli 2012; Marka and Nanna 2021), embryonic axes (Kiranmai et al. 2018; Venkatesh et al. 2022), hypocotyl (Dodo et al. 2008), epicotyl (Qiusheng et al. 2005; Akram et al. 2016), axillary buds (Venkatachalam et al. 1998; Yin et al. 2007), cotyledonary nodes (Vasavirama and Kirti 2012; Lamboro et al. 2022), seedlings, embryonic axes (in-planta) (Patil et al. 2014; Kumar and Kirti 2015), and embryogenic calli (Higgins 2004; Livingstone et al. 2005). Achieving efficient regeneration is a critical and fundamental step to ensure successful plant transformation through tissue culture. For instance, a regeneration protocol was optimized for peanut cultivar N3 and Yu-Hua-14 by testing different concentrations of BAP, TDZ, 2,4-D, and NAA hormones using cotyledonary nodes as explants (Lamboro et al. 2022). The regeneration frequency of this method was 93.4% using 4 mg/L BAP in shooting media and 1 mg/L NAA in rooting media (Lamboro et al. 2022). Another study optimized regeneration for Florunner and New Mexico Valencia A (NMVA) cultivars to obtain direct shoot organogenesis from cotyledonary node (CN) explants, which resulted in 98% regeneration frequency using 6.66 μM (BAP) (1.5 mg/Lit) and 5.37 μM NAA (1 mg/lit) (Hsieh et al. 2017).

The choice of transformation method is a critical factor for achieving successful genetic transformation. For peanut transformation, Agrobacterium tumefacians strains such as LBA4404, GV2260, EHA101, EHA105, GV3101, and C58 (Rohini and Rao 2000; Anuradha et al. 2006; Kim et al. 2009) and particle bombardment (Joshi et al. 2005; Chu et al. 2013) were utilized. Despite variations in transformation frequencies reported in different studies, factors such as the choice of explants, strains, and culture media have been found to impact transformation efficiency, as listed in Table 1. Among these factors, the use of de-embryonated cotyledons has shown promising results, with the highest transformation frequency reported within a range of 58% to 81% (Venkatachalam et al. 1998; Tiwari and Tuli 2012). Further research and optimization of transformation protocols may lead to even higher transformation frequencies in the future. In-vitro transformation of peanut explants exhibits genotypic dependence, as evidenced by various reports (Ozias-Akins and Gill 2001; Anuradha et al. 2006). Therefore, optimizing transformation protocols for each specific peanut genotype, or developing genotype-independent transformation methods, is essential for accelerating peanut crop improvement through genetic transformation. Karthik et al. (2018) have attempted to develop a genotype-independent protocol with enhanced transformation efficiency using peanut cv. CO7. This method was tested in five peanut cultivars, namely CO7, CO6, TMV2, TMV7 and VR13. All cultivars showed variable transformation efficiency ranging from 31.3 to 38.6%.

Table 1.

Reports of optimization of regeneration and transformation in peanut

Genotype	Explant	Method of transformation/Agrobacterium strain	Gene	Selection marker	Transformation efficiency	References
Toalson Florunner	Immature hypocotyl and root shoot axis to generate embryogenic calli	Biolistic bombardment	Uid-A	Hph	1%	(Ozias-Akins et al. 1993)
Tatu (Valencia group)	Leaf (apical half) Leaf (basal half) Embryonated Cotyledons, De-embryonated cotyledon	A281	Uid-A, Bar	npt II and bar	56% 35% 31% 5%	(Mansur et al. 1993)
TAG-24	Leaf	LBA4404	GUS	nptII	7.6%	(Eapen and George 1994)
Florigiant	Embryonic axes with Cotyledon	EHA101	GUS	nptII	9.0%	(McKently et al. 1995)
New Mexico (Valencia)	Leaf sections	EHA105	(uidA)	npt II	0.2–0.3%	(Cheng et al. 1996)
Valencia Florunner Georgia Runner Sunrunner South runner	Leaflets and epicotyls	EHA101, C58	gusA	npt II and hpt	12–36% for leaf lets, 15–42% for epicotyls	(Egnin et al. 1998)
VRI-2 TMV-7	Cotyledons, hypocotyl, epicotyl, axillary bud, immature leaves, cotyledonary nodes	LBA4404	Uid-A	npt II	Cotyledon-40%, Hypocotyl-47%, Cotyledonary node- 58%	(Venkatachalam et al. 1998)
GK7	Leaflets	Microprojectile bombardment	GUS	hph	15%, 36%	(Kim et al. 1999)
Gajah, NC-7	Embryonic axes from Mature seeds	Microprojectile bombardment	luc, uid A	hpt	44% of regenerated plants	(Livingstone and Birch 1999)
TMV-2 JL-24, ICGS-44, ICGV86564	Embryonic axes	LBA4404	Uid-A	npt II	3.3%	(Rohini and Rao 2000)
Tatu	leaflets	Modified electroporation	GUS	nptII	57.1% of regenerated plants	(Padua et al. 2000)
Luhua 9 YueYou 116	Cotyledons of immature Zygotic embryos	Gold particle, helium	Uid-A,	Hph	1.6%	(Deng et al. 2001)
Georgia Runner	Embryonic axis	Microprojectile bombardment	MerApe9	hpt	22 plants	(Yang et al. 2003)
Gerogia green	Mature zygotic embryos	Microprojectile bombardment	EGFP, EYFP	hph	68% co-transformation frequency	(Joshi et al. 2005)
JL-24	Cotyledonary node	GV2260	Promoterless synthetic gus::nptII bifunctional fusion gene	npt II	3.5%	(Anuradha et al. 2006)
-	Leaf	R1000	GUS	nptII	70%	(Kim et al. 2009)
JL-24	De-embryonated cotyledons (DEC)	EHA101, LBA4404	β-Glucuronidase (uidA)	Hpt/nptII	81%	(Tiwari and Tuli 2012)
Georgia Green	Zygotic embryos	Biolistic Bombardment	Ara h1, gfp	Hph	64% of regenerated plants	(Chu et al. 2013)
CO7, CO6, TMV2, TMV7 and VR13	cotyledon with full embryo (in-planta)	EHA105	GusA, Bar, hptII	Bar	31.3%–38.6%	(Karthik et al. 2018)
JL-24	Cotyledonary nodes	EHA105	AtHDG11	nptII	2.8%	(Banavath et al. 2018)
Shanhua 7, Luhua 8 and Huayu 9616	seedling leave pieces	Agrobacterium mediated	∆9-elongase, ∆8-desaturase, ∆5-desaturase, ∆15-desaturase and ∆17-desaturase	nptII	5 plants	(Wang et al. 2019)
ICG 13942	De-embryonated cotyledon	LBA4404	Tcchitinase-I from T. cacao	nptII	63.34%	(Marka and Nanna 2021)
K6	Embryonic axes	EHA105	MuMYB96, MuWRKY3, MuNAC4	nptII	-	(Venkatesh et al. 2022)
Zhonghua-2 (ZH2)	Pollen tube transformation	GV3101	AhBI-1	hpt	50% frequency of positive transformants	(Zhou et al. 2023)
Zhonghua 12 Zhonghua 24 and Huayu 23	Cotyledons, 1-week-old germinated seeds without radicle and hypocotyl	PEG-mediated protoplast transformation, GV3101, K599	DsRed2	Basta	Increased 56.9% to 100%	(Huai et al. 2023)
L14	de-embryonal cotyledons	LBA4404	42 kDa chitinase gene from Trichoderma asperellum SH16	nptII	31.03% of regenerated plants	(Hoa et al. 2023)

Promoters play a crucial role in governing the expression of genes. The CaMV35S promoter is the most employed constitutive promoter for transformation optimization studies and gene expression in transgenic peanut plants. Several tissue-specific or inducible promoters offer elevated gene expression within a particular tissue or at a specific developmental stage and deliver more targeted gene expression, with little to no impact on the final yield. Stress-inducible promoters such as rd29A (Ramu et al. 2016; Banavath et al. 2018) and PSARK (Qin et al. 2011) were used to express stress-responsive genes to develop peanut with improved drought tolerance. Seed-specific promoters Oleosin (Bhatnagar et al. 2010; Yang et al. 2011) and Phaseolin (Chu et al. 2013) were utilized to express specific proteins during seed development. Incorporation of specific reporter genes with the genes of interest facilitates the identification and recovery of positively transformed plants. Reporter genes such as Uid-A, EGFP, EYFP, and DsRed2 reporter genes significantly enhance the recovery of positive peanut transformants during screening (Joshi et al. 2005; Tiwari and Tuli 2012; Huai et al. 2023).

Genetic engineering to develop biotic stress resistance in peanut

Peanut is often exposed to several biotic stresses encompassing fungal, bacterial, and viral pathogens and insect pests, resulting in a significant decrease in yield and quality (Yang et al. 1998). The limited genetic diversity and the complex tetraploid nature of the cultivated peanut gene pool pose significant challenge in developing peanut cultivars with superior quality, high yields, and broad resistance to biotic and abiotic stresses (Pandey et al. 2017). To combat biotic stresses, researchers have expressed genes encoding glucanase, coat proteins, cell wall components, chitinase, oxalate oxidase, bacterial chloroperoxidase, crystal proteins, and pathogen-related genes into peanut plants. Notably, although RNAi-mediated resistance has been effective against RNA viral infections in other crops, this approach has yet to be explored in peanuts.

Insect pests directly consume developing plant parts and pods, and their damage can also attract pathogenic fungi like Aspergillus flavus, which contaminates the crop, leading to reduced yield and compromised quality. Diverse Cry genes found in Bacillus thuringiensis (Bt) bacteria code insecticidal endotoxin crystal proteins (Das et al. 2021). Genetically engineered peanuts with the introduction of CryIA(c) from Bt showed various resistance levels to the lesser cornstalk borer (Singsit et al. 1997). Similarly diverse forms of cry genes like Cry1AcF, and Cry1EC were introduced into peanut showed resistance against insect pest Spodoptera litura (Tiwari et al. 2008, 2011; Keshavareddy et al. 2013), cry1EC + Chi11 against Phaeoisariopsis personata and S. litura (Beena et al. 2008), cry1X against defoliators Helicoverpa armigera and Spodoptera litura (Entoori et al. 2008), cry8Ea1, cry8Ea1 + MARs gene against insect pest Holotrichia parallela (Geng et al. 2012, 2013).

Fungal pathogens cause extensive yield and quality losses in peanut crops. Major fungal pathogens such as Cercospora arachidicola and Phaeoisariopsis personata cause leaf spot or Tikka disease of peanuts, Aspergillus flavus causes aflatoxin contamination, and Sclerotinia minor causes Sclerotinia blight. Commercial peanut genotypes are not equipped with fungal resistance, so an effective approach is needed to develop peanut cultivars with inherent fungal resistance. Chitinases are pathogenesis-related proteins that tend to accumulate in plants as part of their defense response when the plants are exposed to pathogens. These proteins play a crucial role in plant immunity targeting pathogens, such as fungi and certain insects, that contain chitin in their cell walls. Transgenic peanut lines expressing the Chitinase gene from tobacco (Rohini and Rao 2001), rice (Chenault et al. 2002; Prasad et al. 2013; Akram et al. 2016) provided resistance against leaf spot disease caused by C. arachidicola, P. personata. ICG 13942 cultivar has been developed with enhanced resistance against leaf spot and rust diseases by introducing the Tcchitinase-I gene from T. cacao (Marka and Nanna 2021). BjD (Brassica juncea defensin) is found to provide resistance against leaf spot disease caused by P. personata and C. arachidicola (Anuradha et al. 2008). Transgenic peanut plants expressing SniOLP (Solanum nigrum osmotin-like protein) and Rs-AFP2 (Raphanus sativus antifungal protein2) genes (Vasavirama and Kirti 2012), β 1–3 glucanase from arabidopsis (Qiao et al. 2014), glucanase from alfalfa (Chenault et al. 2002), β 1–3 glucanase from tobacco (Sundaresha et al. 2010) and AdSGT1 from A. diogoi (Kumar and Kirti 2015) provided fungal resistance against P. personata. Enhanced resistance against late leaf spot, rust, and A. flavus infection is achieved by introducing the chitinase gene from rice (Prasad et al. 2013). Barley Oxalate oxidase enhances resistance to Sclerotinia blight caused by S. minor in peanuts (Livingstone et al. 2005; Partridge-Telenko et al. 2011).

Peanut is also vulnerable to various viruses, Tomato Spotted Wilt Virus (TSWV), Indian Peanut Clump furoVirus (IPCV), Potato Spindle tuber Viroid (PStV), Tobacco Streak Virus (TSV) causes Peanut Stem Necrosis Disease (PSND), and Bud necrosis disease in peanut (PBND) caused by Peanut Bud Necrosis Virus (PBNV), resulting in significant yield losses (Radhakrishnan et al. 2016; Das et al. 2021). Current management strategies for viral disease include controlling their transmission vectors, such as pests, through pesticides and removing susceptible weed hosts, but these practices are not fully successful. RNAi mediated gene silencing technology has been utilized in various agronomic crops to provide resistance against viruses. Peanut plants expressing the antisense nucleocapsid (N) gene from TSWV showed resistance against TSWV (Yang et al. 1998; Magbanua et al. 2000), while those expressing the PBNV-N nucleocapsid gene exhibited resistance to PBNV (Rao et al. 2013). Similarly, RNAi technology has been applied to provide resistance against TSV, PStV, and IPCV by utilizing genes encoding Coat protein of Tobacco streak virus (TSV-CP) (Mehta et al. 2013; Senthilraja et al. 2018), PStV Coat protein CP2 and CP4 (Higgins et al. 2004), and IPCV-Cp coat protein (Sharma and Anjaiah 2000) respectively.

Genetic engineering for Abiotic stress resistance in peanut

Peanut is cultivated under both irrigated and rain-fed conditions and often face challenges posed by soil salinity and insufficient water conditions that affect their growth and productivity. Furthermore, crops affected by abiotic stresses are more vulnerable to weeds, pests, and diseases, directly contributing to elevated losses and adversely affecting overall productivity. Plants initially respond to these stresses by detecting stress signals and subsequent signal transmission, which ultimately triggers the activation of various physiological and biochemical pathways. In signal transduction, stress signals result in the expression of stress-responsive genes through networks. The crucial stage in enhancing plant stress tolerance revolves around triggering the expression of stress-related genes, a process predominantly controlled by specialized TFs. The activation of abiotic stress-related genes and TFs such as DREB, PDH45, MYB, NAC, mtlD, PAPX, NHX, and WRKY3 in various peanut varieties has led to increased resilience against drought, salinity, elevated temperature, and osmotic stress. Furthermore, the expression of these transgenes can modulate biochemical pathways, resulting in reduced lipid peroxidation, improved photosynthetic efficiency, scavenging of free radicals, controlled transpiration rates, and enhanced water use efficiency (Mallikarjuna et al. 2016). A cis-acting DRE plays a crucial role in regulating the expression of genes responsive to dehydration when plants are exposed to water-deficit stress (Singh and Chandra 2021). TFs DREB1 and DREB2 bind to the promoter region of these dehydration-responsive genes, thus triggering their expression in reaction to various abiotic stresses. Transgenic peanut plants expressing DREB1A under a stress-responsive promoter from the rd29A gene showed improved water use efficiency and enhanced resistance to drought conditions (Bhatnagar-Mathur et al. 2007, 2014; Rana and Mohanty 2012). Similarly, transgenic peanut plants expressing cytokinin biosynthesis AtIPT gene under maturation and stress-inducible promoter PSARK maintained higher photosynthetic rates, stomatal conductance, transpiration, and significantly higher yields than wild-type under water-limited conditions (Qin et al. 2011). The NAC gene family encodes many TFs in plants, providing tolerance to plants against abiotic stresses (Nuruzzaman et al. 2013). Peanut transgenic lines expressing MuNAC4 exhibited enhanced tolerance to drought stress by reducing the oxidative damage to membrane structures, enhancing osmotic adjustment, and balancing the redox potential under water stress (Pandurangaiah et al. 2014).

WRKY TFs regulate several downstream stress-responsive genes and play an essential role in plant biotic and abiotic stress responses. WRKY3TF gene from a drought-adapted horse gram was incorporated into the peanut. The transgenic peanut lines expressing MuWRKY3 showed the up-regulation of stress-responsive HSP, LEA, APX, MIPS, CAT, and SOD genes resulting in increased tolerance to drought stress (Kiranmai et al. 2018). Mannitol is a compatible solute that aids in adjusting cell osmotic balance and can neutralize free hydroxyl radicals generated under different abiotic stress conditions. A bacterial mtlD gene encoding mannitol 1-phosphate dehydrogenase was introduced and overexpressed in peanut cultivar GG 2. Transgenic peanut lines showed elevated levels of mannitol and improved water-deficit stress tolerance (Bhauso et al. 2014). A stress-responsive helicase, PDH45, from pea known for its excellent water-related traits was introduced and overexpressed into a peanut. The resulting transgenic peanut lines exhibited a “stay-green” phenotype, stimulated root growth in response to stress, and improved chlorophyll content that ultimately displayed increased water use efficiency under stress conditions (Manjulatha et al. 2014). Similarly, drought-adaptive traits were pyramided by the simultaneous incorporation of three genes, namely Alfin1, PgHSF4, and PDH45. Transgenic peanut lines expressing these genes showed improved stress tolerance, higher growth, and productivity under drought-stress conditions. Several stress-responsive genes such as HSPs, RBX1, Aldose reductase, LEA5, and PRP2, showed enhanced expression under abiotic stress in transgenic lines (Ramu et al. 2016).

Salinity stress is a major environmental constraint that adversely affects agricultural growth and productivity worldwide. About 6% of the total land is affected by salt worldwide, 22% of which is cultivated land and is unsuitable for growing crops because of contamination with high salt levels (Singh et al. 2014). A high salt level in soil hinders the water uptake by roots from the soil, and within plant cells can be toxic to cellular enzymes. AtNHX1 encodes a vacuolar sodium/proton antiporter that sequesters excess sodium ions out from cytosol into the large intracellular vacuole. The accumulation of sodium ions in vacuoles restores the cytosolic osmolality that favors water uptake through roots and improves water retention in tissues under salt-affected soils. The AtNHX1-expressing peanut plants contained more chlorophyll, maintained higher photosynthetic rates, and increased biomass under salt stress conditions (Asif et al. 2011; Banjara et al. 2012). The AtAVP1 gene encodes an H + pyrophosphatase, which acts as a proton pump in the vacuolar membranes. This process creates a proton gradient across the membrane, serving as the energy source for several secondary transporters located on the vacuolar membranes, including Na⁻/H⁺ antiporters. The transgenic peanut lines expressing AtAVP1 exhibited increased biomass production and higher photosynthesis rates in both drought and saline environments (Qin et al. 2013). Transgenic peanut plants incorporated with SbpAPX of an extreme halophyte Salicornia brachiate exhibited adequate tolerance to oxidative and salt stress conditions (Singh et al. 2014).

Co-expression of three stress-responsive TFs AtDREB2A, AtHB7 and AtABF3 in transgenic peanut plants induced cellular tolerance pathways involved genes like AhRbx1, AhHSP70, AhDIP, AhLea4, AhGlutaredoxin, AhAldehyde reductase, AhProline amino peptidase and AhSerine threonine kinase-like protein. Multigene-expressing plants exhibited improved membrane and chlorophyll stability due to enhanced reactive oxygen species scavenging and osmotic adjustment by proline synthesis under stress. Transgenic lines showed increased tolerance to drought, salinity, oxidative stresses, and total plant biomass compared to wild-type lines (Pruthvi et al. 2014). Another TF, AtNAC2 from Arabidopsis, when expressed in other plants, showed improved drought and salinity tolerance and yield under water-limited conditions (Patil et al. 2014). Three stress-responsive regulatory TFs from horse gram MuMYB96, MuWRKY3 and MuNAC4, were introduced and co-expressed in peanuts. The multigene transgenic peanut lines showed improved antioxidative defense mechanisms and higher maintenance of tissue water status under drought stress. Expression analysis of transgenes and their downstream target genes, KCS6, KCR1, APX3, CSD1, LBD16, and DBP, showed a two- to four-fold increase in transcript levels in multigene transgenic peanut plants under drought stress. This demonstrates that the simultaneous expression of these regulatory genes significantly enhances drought tolerance and overall plant performance in peanuts (Venkatesh et al. 2022).

Genetic engineering for reducing aflatoxins and allergens

Peanut pods are more susceptible to fungal infections as they remain in contact with soil, and warmer conditions further exacerbate the vulnerability of fungal infection and aflatoxin contamination. Aflatoxins are mycotoxins produced by Aspergillus flavus and Aspergillus parasiticus having mutagenic and carcinogenic properties that often contaminate food and food products (Bhatnagar-Mathur et al. 2015). Aflatoxin contamination poses a pervasive challenge across the peanut industry, impacting farmers, consumers, and manufacturers. Genetic engineering methods hold the potential to create peanut varieties with inherent resistance to fungal infections, offering a promising avenue to mitigate aflatoxins contamination issues. Utilizing the GE approach reduced aflatoxin contamination in peanut seeds was obtained by introducing the nonheme chloroperoxidase gene (cpo-p) from Pseudomonas pyrrocinia (Niu et al. 2009). Sharma et al. (2018) reported a reduction in pre-harvest Aspergillus flavus infection by overexpressing two antifungal defensin genes, MsDef1 and MtDef4.2. Further, host-induced gene silencing of two aflM and aflP aflatoxin biosynthetic genes reduced aflatoxin contamination (Sharma et al. 2018). Additional research is imperative to advance the development of peanut varieties resistant to fungal infections and aflatoxin accumulation.

Another significant concern in peanut consumption is the presence of allergenic compounds. The allergenic compounds in peanuts are reported to cause life-threatening health complications that are a major concern for peanut-containing foods and peanut-based industries. There are a total of eighteen peanut allergens (Ara h 1–Ara h 18) identified so far, of which eleven have displayed remarkable potency and belong to the protein superfamily of plant defense proteins, cupin and prolaminin (Finkelman 2010; Zhuang and Dreskin 2013). Ara h 2 and Ara h 6 are major allergens (Flinterman et al. 2007; Koppelman et al. 2010). The proteolytic digestion, exposure to heat, and chemical degradation have proven ineffective against the peanut allergens (Koppelman et al. 2010; Toomer et al. 2013). With advances in GE technologies, the factors responsible for peanut allergy could be modified within plant sources. An attempt was made to silence major allergens Ara h 2 and Ara h 6 in peanuts using RNA interference (RNAi) technology. The crude extract from transgenic peanut seeds showed a significant decrease in IgE binding capacity evaluated by ELISA using sera of patients allergic to peanuts (Chu et al. 2008; Dodo et al. 2008). This suggests that new-era genome editing technologies such as CRISPR-Cas9, and prime editing have more potential and can be utilized to specifically modify the allergen-causing epitopes.

Previous reviews have provided valuable insights, primarily centered on experimental advances and transformation techniques used in genetic engineering to enhance desirable traits in peanut crops (Guo et al. 2023; Krishna et al. 2015; Roy and Sandhu 2024; Singh et al. 2024; Zhou et al. 2023). However, a fully integrated approach that employs CRISPR-Cas9 for peanut crop improvement from genotype selection to producing successful transformants with targeted traits remains unexplored. This review addresses viable genome-editing strategies aimed at enhancing peanut crops resilience to biotic and abiotic stresses, while also identifying future research directions in this field. Additionally, we illustrate recent applications of CRISPR-Cas9, including vector construction and transformation techniques, critical for achieving targeted gene modifications in peanut crops (Fig. 2).

Fig. 2.

Genetic engineering approaches for peanut crop improvement in the post-genomic era. Various genetic engineering strategies that can be employed to enhance the genetic composition of peanut crops. The diagram showcases key steps in the process, including A Stress Induction: Inducing stress in a controlled environment after selecting peanut genotypes showing contrasting stress tolerance and susceptibility traits. B Sample Collection: DNA/RNA/Protein samples are collected and isolated. C Omics Application: Single or multi-omic approaches in identifying candidate genes/proteins/metabolites/enzymes/hormones associated with biotic or abiotic stresses. D List of genes: A few candidate genes that have been used in crop genetic improvement. E Different approaches: The choice of approaches, including RNAi, ZNF, TALENs, CRISPR-Cas9, C/A base-editors, and Prime-editors are presented F Vector Construction: plasmids are constructed depending on the choice of application. G Transformation Techniques: The choice of particle bombardment, Agrobacterium-mediated, Viral-mediated, PEG-mediated, and Nanoparticle-mediated transformation techniques are presented. H Selection and Regeneration: Select successful transformants with desirable traits and regenerate them on selective media into a full plantlet. I Improved Peanut: Collection and evaluation of improved peanut events with desirable characteristics such as stress tolerance or disease resistance

Genome editing for peanut improvement

Advancements in crop transformation and genome editing technologies are paving the way for improving peanuts. Earlier editing techniques like Zinc Finger Nuclease and Meganuclease have not been utilized in peanuts, potentially due to challenges in vector design and construction. TALENs have been utilized to target the fatty acid desaturase 2 (ahFAD2) gene, a key enzyme for catalyzing the conversion of monounsaturated oleic acid into polyunsaturated linoleic acid. Enhancing the concentration of oleic acid has emerged as a primary objective in peanut breeding due to its advantageous effects on health, offering antioxidant properties, lowering blood cholesterol levels, and, along with its consumer and industrial advantages such as flavor, nutritional quality, and extending shelf life (Derbyshire 2014). Until now, most high oleic acid peanut genotypes have originated from spontaneous genetic mutations in both genes. The gene-editing approach promises to induce novel de novo mutations in the ahFAD2 genes. Wen et al. (2018) developed peanut mutant lines with a 42.5–92.5% increase in oleic content by creating mutations in ahFAD2 genes using TALENs. While CRISPR/Cas genome editing is easier to program, it only requires an RNA to guide Cas endonucleases. CRISPR/Cas9 has been applied in peanuts to demonstrate the system's applicability. However, the challenges in the transformation process and low efficiencies have hindered the generation of stable transgenic lines. This highlights the need for further research to improve the efficiency of transformation. A comprehensive list of genome editing investigations in peanuts is outlined in Table 2.

Table 2.

Reports related to Genome editing (ZFN, TALEN, CRISPR) for peanut crop improvement

Gene	Editing tool	Type of mutations	Vector used	Promoter expression	Transformation method/Agrobacterium strain	Purpose	References
AhFAD2	TALENs	Deletion	pCAMBIA1301- TALENs-FAD2	35S	EHA105	twofold increase in the oleic acid content (60–80%)	(Wen et al. 2018)
ahFAD2A/ahFAD2B	CRISPR/Cas9	Transition, Insertion, Transversion	p201B-Cas9	MtU6 promoter	K599	Demonstration of CRISPR/Cas9 mediated mutations in FAD2 gene	(Yuan et al. 2019)
AhNFR1 and AhNFR5	CRISPR/Cas9	InDels	p201G/Cas9	MtU6 promoter	K599	Functional Validation of nodulation genes	(Shu et al. 2020)
Arahy.4E7QKU and Arahy.L4EP3N	CRISPR/Cas9	Indels	pCAMBIA2300-cas9_FatB	AtU6 promoter	EHA105	Improvement of oil quality in peanut	(Tang et al. 2022)
Restoration of gfp	Prime-editing	Site specific Restoration of mutation	pMOD_2515b/pMOD_B2303_pegRNA in pTRANS_100	CmYLCV promoter	PEG mediated Protoplast	Validation of prime editing	(Biswas et al. 2022a)
Allergen Gene Ara h 2	CRISPR/Cas9	Deletion	pTRANS_100	CmYLCV promoter	PEG mediated Protoplast	Functional Validation	(Biswas et al. 2022b)
RY and 2S motif in AhFAD2	CBE (CRISPR/nCas9)	C to T (47 and 59%), C to G (40 and 26%) and C to A (13 and 15%), G to A	pDW3873 and pDW3876	AtU6-26	K599	Demonstration of base editing	(Neelakandan et al. 2022a)
RY and 2S seed protein CRE motif in FAD2 gene	CRISPR/Cas9	Indels	pDW3898 and pDW3899	Arabidopsis U6 promoter	K599, Agrobacterium strain GV3101-mediated calyx tube injection	Functional validation and increase in oleic acid levels	(Neelakandan et al. 2022b)
AhFADH2	CRISPR/Cas9	Indels	pDW3877 and pDW3872	AtU6-26	K599	Demonstration of Improved scaffold plus terminator strategy	(Neelakandan et al. 2022c)
AhFADH2B	CRISPR/Cas9	Insertion	BGK41-Cas9 recombinant vector FAD2B-1	Soyabean U6 promoter	Node injection with Agrobacterium tumefaciens strain GV3101	> 80% oleic acid	(Han et al. 2023)
AhALS2-A and AhALS2-B (Pro197)	CBE	Base editing	pCSGAP01	AtU6 promoter	Microprojectile bombardment	To generate herbicide-resistant peanut plants	(Shi et al. 2023)
AhMULE9A	CRISPR/Cas9	Indels	PHK2-Cas9-U6:sgRNAs	AtU6 promotor	GV3101	Functional validation of AhMULE9A	(Li et al. 2023)
AhKCS1 and AhKCS28	CRISPR/Cas9	Indels	–	–	GV3101	Reduced of VLCFA content	(Huai et al. 2024)

Selecting the right target site is crucial in CRISPR/Cas9 mutagenesis, as efficiency heavily depends on the selected sequence (Janga et al. 2017). Therefore, validating the experimental design in protoplasts or through hairy root generation is critical before generating stable transgenic lines, especially in recalcitrant crops like peanuts. Yuan et al. (2019) demonstrated the application of CRISPR/Cas9 technology in peanut protoplasts and hairy root cultures by creating mutations in the AhFAD2 gene. Shu et al. (2020) validated the functions of AhNFR1 and AhNFR5 nodulation genes utilizing CRISPR/Cas9 technology through a hairy root transformation system in peanuts (Shu et al. 2020). Desired modification can be achieved precisely through base editing or prime editing, which necessitates thorough testing of the designed vectors in protoplasts or hairy roots. Cytosine or adenine base editors directly create precise point mutations in the target region without making double-stranded DNA breaks (DSBs) (Rees and Liu 2018). The application of cytosine base editor was demonstrated in peanuts by targeting the RY and 2S cis-regulatory elements and the coding region of AhFADH2 gene using hairy root transformation (Neelakandan et al. 2022a). Similarly, Neelakandan et al. (2022b) also performed site-specific genome modification of the AhFAD2 gene using the calyx tube injection method. Few T0 seeds exhibited an increase in oleic acid, ranging from 55 to 70%. However, the absence of mutation inheritance in T1 plants indicates that the transformations in T0 seeds were unstable, and the mutations were somatic rather than germline.

Prime editing represents a recently developed, highly accurate editing technology that relies on CRISPR-Cas9. This method utilizes an engineered reverse transcriptase (RT), a catalytically impaired Cas9 endonuclease (nCas9), and a guide RNA specific to prime editing (pegRNA). Notably, prime editing offers a broader spectrum of editing capabilities than base editing, allowing for generating nearly all types of edits (Anzalone et al. 2019). Application of prime editing was also demonstrated in peanut protoplasts by site-specific restoration of the gfp gene (Biswas et al. 2022a). However, it is important to note that stable transgenic plants were not produced in any of the investigations mentioned above, and all are demonstrations either in protoplasts or through the hairy root system or the calyx tube injection method. Only two studies have documented the successful generation of stable transformants in peanuts by applying the CRISPR/Cas9 system. In one study, CRISPR/Cas9 was employed to knock out the AhFAD2B gene in peanuts, resulting in T1 seeds from stably transformed plants having over 80% increased oleic acid content (Han et al. 2023). Another investigation generated herbicide-resistant peanut lines by precisely modifying C to T at the Pro197 position in the AhALS2 gene using CRISPR/nCas9 CBE technology (Shi et al. 2023). Genome editing techniques, notably the CRISPR/Cas9 system, have been effectively demonstrated to manipulate the peanut crop. Enhancing the efficiencies of transformation and CRISPR technologies are crucial objectives for future research to facilitate the development of peanuts with novel traits.

Future prospective of genetic engineering in peanut crop

In the post-genomic era, genetic engineering has emerged as a potential and rapid solution to improve the genetic composition of peanut crops, as illustrated in Fig. 2. Genetic engineering offers solutions to discover and introduce novel traits that can withstand both biotic and abiotic challenges. The process of genetic engineering necessitates a well-optimized regeneration and transformation protocol. While most peanut transformation protocols involve direct organogenesis or somatic embryo formation to generate transgenic plants. However, the rate of somatic embryo formation is significantly lower. Direct organogenesis is a more efficient method for generating transgenic plantlets than somatic embryogenesis. Nevertheless, the success of a transformation system relies on the use of a genotype, suitable explant, strain of Agrobacterium, selection agent, media composition, and growth conditions. Cotyledonary nodes have demonstrated higher responsiveness to organogenesis than other explants (Table 1). Various developmental regulatory (DR) genes were found to play an important role during organogenesis in various plants. TaWOX5, Zmkn1, BBM, WUS, STM, IPT, and GRF4-GIF1 are various developmental regulatory genes, and their utilization in several plants has been reported to enhance transformation efficiency (Yan et al. 2023). The use of DR genes could speed up the transformation process and may help in genetic improvement of peanuts.

The mode of delivery of the vector is also equally important in the transformation process. Apart from Agrobacterium-mediated and biolistic techniques, other delivery methods that offer quicker and less labor-intensive approaches can be explored. Nanoparticle-based DNA delivery system has proven its potential to deliver exogenous biomolecules efficiently (Demirer et al. 2019; Wang et al. 2020). However, this system in peanuts has not been tested yet. Combining such strategies, with new methods of genotype-independent transformation systems can be developed and utilized for accelerating peanut crop improvement. Various selection markers, such as kanamycin, hygromycin, basta, etc., are commonly employed in crop transformation. However, their usage becomes redundant after the transformation process is completed. Furthermore, these selection markers in transgenics pose a challenge to the regulatory processes for their commercial use. Researchers must focus on marker-free genetic approaches to generate peanut transgenics.

As a nutritional crop, peanuts are susceptible to several insect pests specifically lepidopterans, which can cause significant damage to the crop. Crystal proteins (Cry) were found to be toxic in several lepidopteran pests, as these are already utilized in many other crops. Different isoforms of cry genes were inserted in peanuts to develop resistance against several lepidopteran insects. Additionally more novel isoforms of cry genes or other genes are needed to control the insects effectively. Bacteria, viruses, and fungal pathogens cause extensive yield and quality losses in peanut crop. Antisense technology has proven its effective in enhancing resistance against fungal and viral pathogens. Antisense of coat protein gene (Franklin et al. 1993; Higgins et al. 2004; Senthilraja et al. 2018) and nucleocapsid protein gene (Yang et al. 1998; Magbanua et al. 2000; Rao et al. 2013) are utilized for viral resistance. Various other genes used for fungal resistivity are MsDef1, MtDef4.2, AdSGT1, SniOLP, Rs-AFP2, β 1–3 glucanase, BjD, Barley oxalate oxidase, cpo-p and chitinase gene (Qiao et al. 2014; Sharma et al. 2018; Marka and Nanna 2021). Despite the discovery and utilization of various genes for biotic stress resistance in peanuts, there remains a pressing need to identify novel genes and explore innovative approaches. Further research will help in developing more effective and durable resistance strategies against a broader spectrum of pests and pathogens. Researchers across the globe have been working on developing aflatoxin-resistant peanuts, but significant success has not yet been achieved. However the needs for extensive research efforts are necessary to develop aflatoxin-resistant peanuts. Peanuts with broad resistance to Aspergillus spp. could be achieved through a cumulative genetic engineering approach, where researchers can overexpress plant defensive genes and identify and mutate susceptible genes. In addition, antifungal biomolecules and their biosynthetic pathways must be identified and incorporated in peanuts. The Aflatoxin biosynthetic pathway needs to be fully explored, and responsible genes can be silenced or disrupted using RNAi or CRISPR technologies.

Cuticular wax is a protective layer that forms on the outer surface of plant tissues. It is an early adaptive trait that helps plants to resist water stress, harmful UV radiation, and herbivory. To enhance crop plant stress tolerance, researchers must conduct studies investigating the mechanisms of the genes responsible for such physical barriers, like waxes, thick cuticles, and specialized trichomes. Similarly, the mechanism of toxin production in response to various stresses must be discovered (Taiz and Zeiger 2006). The ß-Ketoacyl Co-A Synthase1 (KCS1), catalyzing the rate-limiting step in epicuticular wax biosynthesis, was isolated from the drought-tolerant peanut cultivar K-9 and overexpressed in drought-sensitive peanut cultivar (K-6) under the control of the CaMV35S promoter. Transgenic peanut plants with overexpressed AhKCS1 showed elevated levels of cuticular wax, higher proline content, reduced water loss, decreased MDA content, and lower membrane damage compared to non-transgenic groundnut plants (Lokesh et al. 2019). As an alternate method, the co-expression of multiple stress-responsive TFs offers advantages to modulate the expression of several downstream genes in peanuts. Three genes, Alfin1, PgHSF, and PDH45 from a study (Pruthvi et al. 2014) and AtDREB2A, AtHB7, and AtABF3 genes from another study (Ramu et al. 2016) have been co-expressed in peanut plants to improve abiotic stress tolerance. The co-expression of multiple stress-responsive genes shows great potential for developing peanut varieties that exhibit enhanced tolerance to drought and salt stress. (Pruthvi et al. 2014; Ramu et al. 2016; Venkatesh et al. 2022). Stress-inducible promoters can be employed to drive gene expression, specifically under stress conditions. Employing stress-inducible promoters like rd29A and SARK for expressing of stress-responsive genes represents a superior strategy to reduce the additional burden on plants (Qin et al. 2011; Rana and Mohanty 2012).

Peanut crop has the potential to be genetically engineered for the production of beneficial fatty acids and other valuable natural products like resveratrol and eicosapentaenoic acid (EPA) having therapeutic activities (Halder and Jha 2016; Wang et al. 2019). This can be achieved by introducing a comprehensive set of genes involved in the biosynthetic pathway. A comprehensive understanding of the mechanisms governing oil profiles in peanuts is still required. This knowledge gap represents an opportunity for further research that could be leveraged to modify fatty acid composition and improve oil profiles in peanuts. In peanuts, cytosine base editing technology has been successfully demonstrated by editing the AhFAD2 gene in the hairy root system (Neelakandan et al. 2022a), and CBE is also utilized to create herbicide-resistant stable peanut plants by editing the AhALS2 gene using microprojectile bombardment (Shi et al. 2023). Along with nutritional rich properties, peanuts also contain many allergenic compounds which cause severe allergic reactions in susceptible individuals. Significant efforts have been directed toward reducing these allergens using biotechnological approaches such as RNA interference (RNAi) (Dodo et al. 2008) and CRISPR/Cas9 (Biswas et al. 2022b). While knocking down or fully removing allergenic genes can help reduce allergenicity, this strategy may adversely affect seed composition, as these genes encode essential seed storage proteins. To address this challenge, precise editing of specific epitopes on allergenic proteins offers promising solution. Modifying these binding sites can prevent interaction with human antibodies, reducing allergenicity without disrupting the protein's role in seed development. Prime editing, an advanced genome-editing technique, now enables us to modify these epitope regions with high accuracy, retaining protein functionality while mitigating allergic responses. A hypothetical prime editing strategy to remove allergenicity in peanuts is illustrated in Fig. 3. Prime editing for restoring the gfp gene has already been demonstrated in the peanut protoplast system (Biswas et al. 2022a). However, no reports are available for using prime editing in peanuts to develop stable edited plants. RNA interference (RNAi), CRISPR/Cas9, base editing, and prime editing are cutting-edge technologies, and most of their applications have been demonstrated in peanut protoplast and hairy root systems. Other genome editing technologies have not yet been utilized to generate fully developed transgenic peanut plants and products except cytosine base editing. Omics approaches provide immense data and can be applied to peanut crops to identify SNPs and negative and positive regulators in various stresses. Further, CRISPR/Cas technology can be employed to validate and modify these genes, contributing to improving peanut crops.

Fig. 3.

A hypothetical prime editing strategy to reduce/eliminate allergenicity in peanuts. The process of prime editing to reduce peanut allergenicity by targeting the Ara h 2 protein, one of the major allergenic storage proteins. A Peanut Allergens: Displays common peanut allergens, including Ara h 1, Ara h 2, Ara h 3, Ara h 5, Ara h 6, and Ara h 8. B Prime editing: Illustrates the Prime editing process, where alterations are made at the active site of Ara h 2's DNA sequence, leading to modified mRNA and subsequently altered protein structure. C Response of the immune system in the body: This section depicts the response of immune cells to an unmodified peanut allergen (Ara h 2), showing how sensitization leads to histamine release and allergic responses. Prime-edited peanuts, with altered allergenic protein, can potentially evade immune recognition, thus preventing allergic reactions

Conclusions

Genetic engineering has emerged as a transformative approach for peanut crop improvement, offering targeted solutions to complex challenges in cultivation and consumption. By leveraging advanced transformation techniques, particularly Agrobacterium-mediated methods, researchers can develop peanut varieties with enhanced biotic and abiotic stress resistance, improved nutritional profiles, and reduced allergenicity. Transforming currently cultivated genotypes can help farmers mitigate yield losses due to stresses. Emerging genome editing technologies like CRISPR/Cas9 provide unprecedented precision in genetic modifications, opening new avenues for crop improvement. However, challenges remain in optimizing genotype-independent transformation protocols, ensuring genetic stability, and addressing regulatory concerns. As research progresses, the continued integration of genetic engineering with traditional breeding approaches holds immense potential for developing peanut varieties that offer improved yield, resilience, and nutritional value, ultimately benefiting farmers, consumers, and the global agricultural industry.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.