Antisense RNA (asRNA) Technology: The Concept and Applications in Crop Improvement and Sustainable Agriculture

doi:10.21203/rs.3.rs-2958048/v1

Download PDF

Research Article

Antisense RNA (asRNA) Technology: The Concept and Applications in Crop Improvement and Sustainable Agriculture

https://doi.org/10.21203/rs.3.rs-2958048/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 27 Sep, 2023

Read the published version in Molecular Biology Reports →

You are reading this latest preprint version

Antisense RNA (asRNA) technology is a method used to silence genes and inhibit their expression. Gene function relies on expression, which follows the central dogma of molecular biology. The use of asRNA can regulate gene expression by targeting specific mRNAs, which can result in changes in phenotype, disease resistance, and other traits associated with protein expression profiles. This technology uses short, single-stranded oligonucleotide strands that are complementary to the targeted mRNA. Manipulating and regulating protein expression during its translation can either knock out or knock down the expression of a gene of interest. Therefore, functional genomics can benefit from this technology since it allows for the regulation of protein expression. In this review, we discuss the concept, and applications of asRNA technology which include delaying ripening, prolonging shelf life, biofortification, and increasing biotic and abiotic resistance among others in crop improvement and sustainable agriculture.

Antisense RNA

gene silencing

translation

gapmer

antisense oligonucleotide

The world's population is rapidly increasing, and there is a need to improve food crop production and productivity to meet the growing demand (Godfray et al. 2010; Saurabh et al. 2014; Rajam 2020; Rajput et al. 2021). Traditional plant breeding has been used since the domestication of crops to enhance productivity, environmental adaptation, and quality (Miflin 2000; Animasaun et al. 2020). Today, both conventional and non-conventional breeding methods, including molecular breeding, crop gene editing, mutation breeding, tissue culture, hybridization, antisense technology, and crop genetic transformation, are utilized to develop improved crop varieties. However, the non-conventional methods are faster and more precise than conventional methods (Auer and Frederick 2009; Wang et al. 2023). Thus, advances in biotechnology and genetic engineering tools have made crop improvement more convenient and less cumbersome (Lemmon et al. 2018; Tilahun et al. 2021; Che et al. 2022), even in the production of clean energies (Yan et al. 2020).

Since the discovery of small interfering RNA (siRNA), long noncoding RNA (lncRNA), microRNA (miRNA), and PIWI-interacting RNA (piRNA), typically non-coding RNAs (ncRNA), they have generated popular research topics. Antisense RNA (asRNA) which is a form of ncRNA participates in the regulation of the genetic activity of the chromosome structures, transcription and translation as well as the RNA and protein stabilities (Rusk 2015: Xu et al. 2018). The asRNA is a unique form of the small noncoding molecule of the DNA transcript that complements specific mRNA. Antisense RNA technology involves manipulating the RNA molecule to prevent the expression of a specific gene. Though previously thought to be a “junk RNA” (Boland 2017), asRNA possesses immense potential for intercellular gene interaction and can be a veritable tool for gene silencing. In some cases, asRNA, hairpin (HP) as well as hairpin antisense (HPAS) construct was used to improve plant (Nguyen and Shanklin 2008).

Gene expression involves two main steps: transcription, in which the DNA sequence serves as a template to create an mRNA molecule, and translation, in which the mRNA is used as a template to synthesize a protein (Clancy and Brown 2008; Gupta et al. 2011). The genes in DNA provide the instructions for making proteins, which are essential for the functions of the cell. During translation, the mRNA sequence is translated into a specific sequence of amino acids, which form a polypeptide chain that folds into a functional protein (Barry 2007; Clancy and Brown 2008). The genetic code is based on a triplet code, where each codon consisting of three nucleotides specifies an amino acid. Several enzymes, including polymerase, exonuclease, ligase, etc., work in concert to unwind the DNA double helix and match RNA base pairs with the DNA bases to produce the RNA copy of the DNA. The produced mRNA molecule is immature, heterogeneous nuclear RNA (hnRNA) (Clancy and Brown 2008; Kumar 2010; Lemmon et al. 2018) that is not yet for gene expression. The introns of the pre-mRNA must be spliced out and shielded through a 5' cap and poly-A tail addition to protect the mRNA from degradation. (Gupta et al. 2011). The pre-mRNA is subsequently moved out of the nucleus into the cytoplasm where it is transformed into mRNA in preparation for the next step (translation). The translation occurs in the cytoplasm, the mRNA binds to the ribosomes which are responsible for protein synthesis. The description of the basic workflow of the central dogma is illustrated in Fig. 1.

Gene silencing is an effective molecular procedure that inhibits the expression of a specific gene, and it serves as a regulatory mechanism for gene expression, determining cell fate and metabolic activities during an organism's lifespan. Gene silencing eliminates the effect of a gene by inhibiting its expression (Dash et al. 2015; El-Sappah et al. 2021; Woodward 2022). The presence of double-stranded RNA (dsRNA) in the cytoplasm is the primary factor that induces gene silencing (Fire et al. 1998; Mu et al. 2018). In plants, gene silencing is critical for protection against abnormal RNA, which can be fatal if it persists in the RNA pool of a cell (Dunoyer et al. 2006). This is achieved through nonsense-mediated mRNA decay (NMD) which could also be used for antisense therapeutic purposes (El-Sappah et al. 2021; Woodward 2022).

Following the central dogma of molecular genetics, gene silencing in plants occurs in two stages: transcriptional gene silencing (TGS) and post-transcriptional gene silencing (PTGS) (Kelly and Aramayo 2007). Transcriptional gene silencing occurs in the nucleus and involves blocking the binding of the transcriptional system to the promoter region, thus inhibiting transcription. PTGS, on the other hand, occurs in the cytoplasm and targets and degrades or blocks mRNA transcripts of specific genes (El-Sappah et al. 2021). Examples of TGS techniques include RNA-directed-DNA methylation (RdDM), paramutation, transposon silencing, genomic imprinting, transgene silencing, and position effect whereas Examples of PTGS techniques include RNA interference (RNAi), antisense RNA (asRNA), clustered regularly interspaced short palindromic repeats (CRISPR/Cas9), and non-coding RNA (NMD) (Wakimoto 1998; Vaucheret et al. 2001; Zebec et al. 2016). Small molecular RNAs such as the asRNA could induce the silencing of a sequence-specific gene by inhibiting the transcription or translation process. Consequently, post-transcriptional gene silencing (PTGS) has been reported in various organisms (Matzkke et al. 2001), and in some cases, the phenomenal suppression of the expressions for both and the homologous endogenous genes was observed (Campbell 2005).

Antisense RNA technology involves modifying RNA molecules to create a complimentary copy of the target messenger RNA (mRNA) in a reverse orientation. This complimentary copy, also called antisense RNA, can bind to the target mRNA, and regulate or inhibit the expression of the corresponding protein (Gupta et al. 2011; Tiwari et al. 2014). Antisense RNA exists in the form of antisense oligonucleotides (ASO/AS-ONs), single-stranded deoxyribonucleotides designed to complement the target mRNA (see Fig. 2). These AS-ONs are usually between 15 and 30 nucleotides in length, and they can be used to effectively regulate gene expression (Crooke 2004; Xu et al. 2018). Zamecnik and Stephenson (1978) were the first scientists to propose the use of small oligonucleotides (ODNs) for targeted gene expression inhibition. Their studies demonstrated that a 13-ODN that complemented the terminally repeated sequences in Rous sarcoma virus (RSV) long terminal repeat (LTR) could prevent Rous sarcoma virus translation in a cell-free system, as well as viral replication in cultured cells in vitro. As time passed, researchers began to fully grasp the potential of antisense-mediated gene regulation.

In the early 1980s, the automation of ODN synthesis made it easy to obtain ODNs of any sequence and evaluate their effectiveness in blocking gene expression via antisense base pairing (Scherer and Rossi 2003; Mo et al. 2012). Antisense oligonucleotides are designed to bind with targeted mRNA via Watson-Crick (1953) base pairing, which involves C-G and A-T. The "sense strand" refers to the original sequence of the DNA or RNA molecule, while the "antisense strand" refers to the complementary sequence of the DNA or RNA molecules (Gupta et al., 2011). The process of hybridizing antisense nucleic acid sequences with a particular mRNA sequence can disrupt the normal processing of genetic information within a cell through various mechanisms. This disruption is either a "knockdown" or a "knockout," depending on whether the information is partially, or completely removed. Thus, the gene function can be determined (Walder and Walder 1988; Mol et al. 1990).

Antisense technology is one of the novel and convenient techniques that crop breeders use to develop and improve crop varieties. Among the tools available to plant biotechnologists, the antisense RNA strategy is especially potent. This approach allows for the targeted reduction of specific gene expressions, resulting in the production of plants with modified traits. It is therefore likely to inhibit characteristics that are deleterious, creating a novel plant mutant for contemporary applications in plant breeding (Bird and Ray 1991; Tilahun et al. 2021). In addition, antisense technologies are employed in functional genomics (Mo et al. 2012).

Solid-phase and enzymatic methods are used to synthesize antisense oligonucleotides (Zaramella et al. 2004). The enzymatic method involves the use of DNA-dependent RNA polymerase enzymes such as sp6, T7 and T3 enzymes. These enzymes catalyze the synthesis of RNA from the 5´- 3´ direction, using either single-stranded or double-stranded DNA as templates. These occur downstream of the DNA template (Ji et al. 2017). In a clearer term, during transcription, the template or non-coding or antisense strand of the DNA is used to create a matching mRNA strand. This mRNA strand is identical in sequence to the coding strand of DNA, except that uracil replaces thymine (Lackner and Bahler 2008). So, the sense/non-template strand of the DNA in the 5´- 3´ direction is transcribed to generate the complementary mRNA strand also known as the antisense RNA (David and Nanette 2016) (see Fig. 2). One can accomplish this through in vitro transcription, which involves using extremely specific DNA-dependent RNA polymerases derived from bacteriophages, such as sp6, T7, and T3 polymerases (Ji et al. 2017).

Solid-phase synthesis involves the sequential addition of nucleotides to a solid support, such as a resin. In this method, the first nucleotide's 3' end is covalently attached to a polymeric support, allowing the oligonucleotide chain to grow on the support. This process uses phosphonamidite as building blocks, which are obtained from protected 2'-deoxynucleosides (dA, dC, dG, and dT), ribonucleosides (A, C, G, and U), or chemically modified nucleosides like LNA or BNA. The building blocks are coupled sequentially to the growing oligonucleotide chain in the desired order to obtain the desired oligonucleotide product (Reese 2005; Tram et al. 2007).

Antisense technology works on the principle of complementary base pairing between antisense oligonucleotide sequence and its corresponding sense RNA strand. This binding impedes translation, the ultimate step in protein synthesis, by blocking the sense RNA strand from participating in the process. The messenger RNA (mRNA) is responsible for transmitting genetic information from the DNA to other cellular components involved in protein production. To put it simply, antisense technology uses complementary base pairing to halt protein production by interfering with mRNA function (Gupta et al. 2011). The antisense approach aims to decrease the expression of a specific molecular target. This is accomplished by triggering the activity of RNase H endonuclease, which cleaves the RNA duplex formed between the antisense RNA and the mRNA of the target gene in the cytoplasm. This cleavage leads to a reduction in protein synthesis and a significant decrease in the translation of the target gene (Godfrey et al. 2017). The primary strategy for AS-ONs (gapmers) is to trigger RNase H activation, which cleaves the mRNA's RNA portion and the antisense RNA duplex, resulting in the degradation of the target mRNA. Furthermore, AS-ONs that do not cause RNase H cleavage can impede translation by physically obstructing the ribosome, known as steric blockers (see Fig. 3a and 3b). When targeting the 5´-terminus with AS-ONs, it is possible to prevent the binding and assembly of the translation machinery. This approach, known as the antisense strategy, is an effective means of selectively manipulating gene expression in plants (Bird and Ray 1991; Jens 2003).

Although the precise process by which mRNA translation is thwarted is not well comprehended, it is believed that the formation of a duplex structure may cause a rapid breakdown of RNA, hinder nuclear processing, obstruct export to the cytoplasm, or impede translation (Gupta et al. 2011; Xu et al. 2018). Since asRNA like other forms of non-coding RNA is a product of dsRNA, a ribonuclease called DICER or Dicer-like enzyme (DCL) catalysis the cleavage (Pare and Hobman 2007) and the process may in addition to the presence of small ncRNA involve the RNA-induced silencing complex- RISC (Wilson and Doudna 2013), and the Argonaute proteins- AGOs (Riley et al. 2012). Essentially, the concept involves introducing a short RNA or DNA molecule into a cell that is complementary to the mRNA produced by a specific gene. By taking advantage of the unique specificity of complementary base pairing as explained by Watson and Crick in 1953, the oligonucleotide can specifically bind to the target mRNA. Consequently, the introduction of an antisense oligonucleotide (ASO) complementary to the mRNA will result in the specific inhibition of protein synthesis by the targeted gene through either cleavage or blockage. Usually, antisense RNA technology involves the overture of a molecular construct with a target gene replicated downstream to a promoter in an inverse orientation promoting the synthesis of antisense RNA complementary to the targeted gene. The duplex formed by the gene and the asRNA complement is subsequently cleaved by a Dicer into short RNA (siRNA) fragments which are integrated into a multi-protein molecule of RNA Induced Silencing Complex (RISC). When RISC is activated, one strand of the siRNA usually refers to as the” passenger” becomes degenerated while the other strand (guide) sticks with the complex (Matzke and Birchler 2005). The activation of RISC ensures specific annealing to the complementary target RNA and the Argonaute, a catalytic component of the RISC, thereby causing the degrading of the target mRNA.

The approach encompasses using ASO to bind to and inhibit follows these assumptions: Blocking RNA splicing, hastening degradation of the RNA molecules, preventing introns from being spliced out of the mRNA, impeding the exportation of mRNA into the cytoplasm, hindering translation, the duplex formation in RNA (see Fig. 4); inhibition of 5′ cap formation, alteration of the splicing process (splice-switching), and steric hindrance of ribosomal activity. The ASO can be directed towards either the initiation codon of methionine (AUG) to hinder translation or splice sites to obstruct splicing (Gupta et al. 2011; Di Fusco et al. 2019). The way ASO works seems to depend on two factors: its sequence and chemistry. The sequence determines the RNA section where the ASO will attach itself, while the chemistry controls whether it will cause RNAse H cleavage or steric blockage (Eric and Cambridge 2018). Post-transcriptional processing of RNAs (pre-mRNA) is nuclear localized whereby the RNA undergoes various modifications, such as the addition of a 5´ cap, splicing, and polyadenylation at the 3´ regions. RNA processing regulation is an effective mechanism for controlling gene expression, and oligonucleotides can be used to achieve this (Dean and Bennett 2003; Dunoyer et al. 2006).

Antisense oligonucleotide (ASO) may directly or indirectly be delivered into the cell. Direct insertion involves the use of a gene gun while the indirect method may be through Agrobacterium tumefaciens, viral-mediated asRNA transfer or by infiltration of ASO into a cell. The indirect method through Agrobacterium is effective in downregulating the quantity of a specific protein (Gelvin 2003; Aregawi et al. 2022). Among the earliest studies involving the indirect approach in plants aimed to compare the effectiveness of sense, antisense, or dsRNA in generating RNA-mediated virus resistance through post-transcriptional gene silencing (PTGS) in Nicotiana tabacum (tobacco) (Waterhouse et al. 1998). Particle bombardment through the gene gun is an effective method of genetically transforming crops such as cereals (Hamada et al. 2018). It is particularly useful for introducing multiple genes and for transforming plants that are difficult to transform through agro-infection. In a recent study, sorghum was transformed using shoot meristem explants and tungsten particles coated with the CYP79A1 antisense RNA construct, which were delivered using helium gas. After bombardment, the explants were cultured on an induction medium and then selected for further study (Pandey et al. 2019).

Applications of antisense RNA technology in crop improvement

Antisense RNA technology is applied in crop improvement in the following areas: Fruit ripening, enhanced shelf-life, reduction of lignin content, enhancement of nutritional qualities, bacterial and virus resistance, altered flower colour, etc. (Le and Wang 2011; Tiwari et al. 2014). Although other forms of antisense technologies such as RNAi, siRNA and miRNA have been widely applied in modifying and improving crops for various purposes, the use of asRNA is gaining more acceptance (Xu et al. 2018; Tilahun et al. 2021; Sinha et al. 2023). Consequently, crops of high economic and cultural significance have been improved using antisense-RNA technology (Table 1), and many more in the nearest decade, as more attention and research interest are geared towards the applications of the antisense-RNA for crop improvement. Furthermore, small non-coding RNA has been employed as a biological tool to investigate gene function in vitro and in vivo and in regulating genes in crop plants in a specific manner without affecting the expression of other genes, consequently increasing their productivity (Kumar et al. 2012). Meanwhile, some of the areas where the techniques have been successfully applied are elucidated below.

Table 1

Some of the crops improved using antisense RNA technology.
SN	Crop	Purpose	Improved traits	Targeted gene(s)	References
1	Tobacco	Biotic resistance	Tobacco mosaic virus resistance	CP	Powell et al., 1989
2	Tomato	Enhanced shelf life Biotic resistance	Delay fruit ripening Virus resistance	1-aminocyclopropane-1-carboxylate (ACC) oxidase Polygalacturonase (PG) gene AL1-coding gene of tomato golden mosaic virus TGMV	Oeller et al. 1991 Day et al., 1991
3	Potato	Biotic resistance Quality improvement Quality improvement	Potato virus resistance Reduced discoloration of tuber after bruising Amylose-free starch	PVX Granule-bound starch synthase (GBSS).	Bachem et al., 1994. Salehuzzaman et al., 1993.
4	Wheat	Quality improvement	Amylose enhancement	Sbe2a	Sestil et al., 2010
5	Grass pea	Quality improvement	Low neurotoxin (β-ODAP)	CoA Synthase	Kumar et al., 2011
6	Cassava	Quality improvement	Hydrogen cyanide (HC) reduction	CYPTGA1	Pandey et al. (2019), Siritungam and Sayre, 2003
		Biotic resistance	Resistance to cassava mosaic virus	AC1, AC2, AC3	Zhang et al., 2005
7	Petunia	Quality improvement	Change in flower colors.	dihydroflavonol-4- reductase	Van der Krol et al., 1988
8	Torenia	Quality improvement	Change in flower colors.	dihydroflavonol-4- reductase	Aida et al., 2000
9	Peanut	Quality improvement	Removal of substances that causes allergy.	Ara h 2 protein-coding gene	Dodo et al., 2008.
10	Mustard	Quality improvement	Oil quality	FAD2	Sivaraman et al., 2004
11	Soybean	Quality improvement	Oil quality	FAD2-1	Zhang et al., 2014
12	Rice	Quality improvement	Increase grain storability	Lipooxygenase (LOX3)	Xu et al., 2014
13	Cotton	Biotic resistance	Resistance to cotton leaf curl virus (CLCuV)	βC1 gene	Sohrab et al., 2016
14	Sorghum	Quality improvement	Reduction of cyanogenic glycoside	CYP79A1	Pandey et al., 2019
15	Maize	Quality improvement	Protein enrichment	22-KD	Frizzi et al., 2010
List of Figures and Legends

Delay ripening and enhancement of shelf life in fruits.

Due to their natural properties and composition, fruits and vegetables are more susceptible to spoiling than cereals. As a result, a huge amount of fruits and vegetables produced are wasted due to spoilage, resulting in inedible waste. Therefore, it is necessary to increase the shelf life of fruits and vegetables to reduce deterioration and spoilage, which would help to minimize horticultural losses. This is an important agronomic trait that should be prioritized. Polygalacturonase (PG) is a crucial tomato cell wall enzyme that is responsible for fruit softening (Rajput et al. 2021) and it plays a significant role in modifying the pectin fraction of the cell wall during ripening. By expressing antisense RNA, plants with lower levels of PG activity have been used to better understand the enzyme's function (Tuck et al. 2016). Polygalacturonase antisense genes were employed, coupled with CAMV 35S promoter to enhance the shelf life of tomatoes via agrobacterium-mediated transformation (Saurabh et al. 2014; Rajput et al. 2021). Ethylene regulates the timing of maturation and extends the preservation of fruits by regulating the expression of genes involved in metabolic processes (Kim et al. 2015). To delay the ripening of the fruit, the expression of the rate-limiting enzymes in the ethylene biosynthetic pathways can be inhibited by using antisense RNA of 1-aminocyclopropane-1-carboxylate (ACC) oxidase from tomato (Oeller et al. 1991; Gupta et al. 2013). Xiong et al. (2005) utilized antisense technology to subdue the expression of the ACC oxidase gene in tomatoes which is associated with ethylene synthesis. The approach led to the extension of the shelf life of the tomatoes, as demonstrated by a reduced ethylene production rate in the ripened fruits of the transgenic plants. Specifically, the introduction of an antisense RNA unit blocked the expression of the gene, resulting in significant inhibition of ethylene production and consequently a longer shelf life for the tomatoes. The use of the antisense technique is promising in suppressing various pathways in plant species. In the case of tomatoes, transgenic varieties were created by introducing an antisense RNA segment of the TOMSSF gene under the regulation of the cauliflower mosaic virus 35S promoter. This resulted in decreased activity of sucrose synthase (SuSy) in the fruits of the transgenic tomatoes (Saurabh et al. 2014; Rajput et al. 2021) and caused a delay in fruit ripening and this approach may be extended to other economical fruits like peas, mangos, banana, apples, and grapes where uncontrolled ripening may cause deterioration and high economic loss.

Virus resistance

Plant viruses are a major factor in reducing crop productivity and yield, as they can affect the quality of crops (Luckanagul et al. 2014). Antisense RNA is effective in creating resistance to plant viruses, as the antisense sequence impedes RNA biosynthesis. Day et al. (1991) reported that antisense RNA can prevent DNA viruses, the antisense DNA of the AL1-coding gene from the geminivirus tomato golden mosaic virus (TGMV) was effective in suppressing TGMV replication. Pathogen-derived resistance (PDR) is a powerful approach to combat virus infections in plants and has been successfully used to engineer virus-resistant plants (Simón-Mateo and García, 2011). Inverted repeats of antisense oligonucleotide of the coat protein (CP) gene of the Tobacco Streak Virus (TSV) have been introduced as an effective and reliable strategy for developing economically important crops that are resistant to TSV (Pradeep et al. 2012). Tomato varieties that are resistant to Potato Spindle Tuber Viroid (PSTVd) were produced through antisense RNA technology (Schwind et al. 2009). Furthermore, in 1989, Powell et al. reported the development of a tobacco variety that is resistant to tobacco mosaic virus (TMV) by using the asRNA technology to modify the CP gene which conferred resistance to TMV.

Developing bacterial-resistant crops

Bacterial diseases pose a significant challenge to field crops, such as tomatoes, soybeans, bananas, etc. due to their fast spread and difficult control. Therefore, it is essential to develop bacterial-resistant varieties of food crops to minimize infections, reduce yield loss, and lower production costs. One promising approach is the use of asRNA to enhance bacterial resistance. In a study by Escobar et al. (2001), silencing two bacterial genes (iaaM and ipt) effectively reduced the production of crown gall tumours caused by Agrobacterium tumefaciens in Arabidopsis to nearly zero. This finding suggests that it may be possible to engineer resistance to crown gall disease in trees, ornamental plants, and even food crops (Saurabh et al. 2014).

Pest resistance

Insect pests are responsible for significant losses in crop yield and the cost of insecticides, with an estimated 20–40% reduction in yield. Despite the use of insecticides, pests have developed resistance, which is exacerbated by the high amount of synthetic pesticides used annually (Ferry et al. 2006; Gordon and Waterhouse 2007; Cagliari et al. 2019). The global expenditure on pesticides is nearly $56 billion, with almost 6 billion pounds of chemicals used in attempts to reduce pest damage (Atwood and Paisley-Jones 2017). However, recent research has shown that unmodified antisense DNA oligonucleotides derived from the RING domain of the LdMNPV IAP-3 gene can cause high mortality rates among gipsy moth larvae, both infected and uninfected with LdMNPV (Oberemok et al. 2019). These oligonucleotides, known as oligoRINGs, function as an antisense RNase H-dependent oligonucleotide, causing degradation of the target mRNA for the host IAP-Z gene. This results in downregulated expression of the target protein, prompting cell apoptosis and larval senescence. The insecticidal effect of oligoRING on LdMNPV-free gipsy moth larvae is believed to be due to interference with the expression of the host IAP-Z gene.

Reduction of linamarin in cassava

Linamarin is a cyanogenic glycoside that is naturally present in many crops, including cassava and lima beans. It can release toxic hydrogen cyanide upon hydrolysis, which poses a health risk to humans and animals that consume these crops. Antisense RNA technology has been employed to reduce linamarin in cassava by downregulating the expression of the cytochrome P450 genes (CYP79D1 and CYP79D2), which catalyzes the first step in linamarin synthesis. The linamarin content in the transgenic crop was reduced by 99% (Siritungam and Sayre 2003).

Reduction of cyanogenic glycoside in sorghum

A major hindrance to the utilization of sorghum forage is the production of cyanogenic glycoside in its leaves and stem, which can be fatal for cattle feeding on it during the pre-flowering stage. To address this, Pandey et al. (2019), isolated and cloned CYP79A1 cDNA in an antisense orientation, driven by the rice Act1 promoter. The resulting construct led to a significant reduction in hydrogen cyanide (HCN) levels in transgenic plants, thus confirming the efficacy of this approach in reducing HCN levels in forage sorghum and making it safer for cattle consumption.

Reduction of lignin content in jute

Flax and jute are important economic crops to the textile and paper industries. However, the linen (textile) and paper industries are currently experiencing a shortage of high-quality flax fibers and bast fibers from jute species due to high lignin content which reduces the qualities of the products made from these plants. Excessive levels of lignin make the fibers rigid and brittle, affecting their textile properties. Moreover, the process of removing lignin produces wastewater that causes severe environmental pollution. Additionally, the high quantity of lignin in commercial jute and flax varieties increases the cost of pulp and fiber production for high-quality textiles and paper. Therefore, there is a need for the development of varieties of jute and flax with low lignin content using asRNA technology or any other molecular tool to address the problem at its root (Xie et al. 2020).

Antisense RNA technology can be utilized to reduce the amount of a crucial enzyme involved in the biosynthesis of lignin in Corchorus species, specifically C. capsularis and C. olitorius, as well as in flax. The enzyme 4-coumarate: CoA ligase (4-Cl) is a vital enzyme involved in the preliminary stages of lignin biosynthesis, along with other enzymes such as phenylalanine ammonialyase (PAL) and cinnamate 4-hydroxylase (C4H). Given the availability of the 4-Cl gene sequence, transgenic varieties of jute and flax can be developed to express the antisense RNA (asRNA) construct that downregulates the 4-Cl mRNA level, which in turn reduces lignin production. Therefore, asRNA technology has the potential to become a potent molecular tool that can generate jute and flax varieties with low lignin content. This would make it easier, more environmentally friendly, and cost-effective to process the fiber for producing various valuable commodities like high-quality paper and cloth (Tanmoy et al. 2014).

Altered colour/scent of flowers.

The demand for economically important flowers like lotus, rose, tulip, petunia, orchids, and poppies with different colors and scents has increased for decoration and fragrance purposes. To improve the quality of these flowers, a gene-silencing approach may be considered. Studies by Seitz et al. (2007) and Nakatsuka et al. (2010) suggest that modification of flower color can be achieved by reducing the accumulation of polyacrylate anthocyanins through asRNA. In a similar vein, Nishihara et al. (2005) found that gene silencing of chalcone isomerase (CHI) in tobacco resulted in reduced pigmentation and altered flavonoid components in flower petals. The study also revealed that CHI plays a significant role in the cyclization reaction from chalcone to flavanone, as evidenced by the accumulation of elevated levels of chalcone in yellow-colored pollen (Saurabh et al. 2014).

Enhancement of nutritional value and quality

One of the goals of plant breeding is to improve the nutritional quality of crops. Antisense RNA technology can be utilized to achieve this objective, as demonstrated in a study by Tilahun et al. (2021). Specifically, antisense RNA technology has been used to enhance the protein quality of maize by targeting seed storage proteins (SSPs), such as Zein proteins which are endosperm-specific proteins in maize, a major staple food worldwide (Zhang et al. 2015). Zein proteins account for 60% of corn proteins, but they lack essential amino acids such as lysine and tryptophan (Frizzi et al. 2010). To produce high-lysine maize variants, RNA silencing has been successfully employed in maize to reduce the expression of 22-KD (α-zeins subfamily) maize zein proteins (Esen 1987; Coleman and Larkins 1999). Decreasing the content of zein proteins through antisense RNA technology leads to the production of maize with higher levels of lysine and tryptophan (Frizzi et al. 2010).

Inducement or restoration of male sterility

In hybridization and plant breeding, male sterility is a crucial factor for purity. Application of antisense RNA in inducing male sterility in self-pollinated crops or restoring the reproductive capacity of the male plant is an important adventure for a successful breeding programme of bisexual plants. Suppression of the mutator S gene (MutS) homolog also known as the Msh1 by preventing its expression in tobacco resulted in cytoplasmic male sterility due to the remarkable mutation of the mitochondria DNA (Sandhu et al., 2007). Some reports demonstrated the use of antisense RNA to produce transgenic plants with induced male sterility or the restoration of the productive ability of once-sterile male plants (Nizampatnam and Kumar 2011). Also, about three decades ago, while working on transgenic petunia plants, van der Meer et al. (1992) declared that antisense RNA of chalcone synthase (CHS) prevents the biosynthesis of flavonoids and covertly caused male sterility of the plant. They summarily remarked that antisense RNA could be utilised by breeders to either induced or restore male sterility in plants and this will give a wider horizon in the genetic manipulation of such plants.

Challenges

One of the key drawbacks of antisense RNA technology is the challenge of effective intracellular delivery of the ASO. The ASO could bind to a non-targeted mRNA, thus the site selection in the mRNA is of great concern (Gupta et al. 2011). Therefore, antisense RMA is yet to be perfected, although the utility of small RNAs as mediators in gene silencing is of immense potential, some of the issues and controversies surrounding its use still need to be addressed with a more convincing approach (Tilahun et al. 2021). There is a need for more comprehensive research to improve the technology and develop an efficient delivery system that will enable the asRNA to bind its targeted mRNA sequence and site without any escape or off-target (Singh et al. 2020). The off-target effect could lead to false experimental inferences and interpretations, and negatively impact or alter the phenotypical expressions of the modified plants which may result in undesirable pleiotropic changes in plant morphology and development. In addition, there is a concern that the asRNA-mediated gene silencing requires specific homology between the asRNA and the targeted mRNA. Thus, long antisense oligonucleotides that could specify a unique mRNA may equally direct the RNase-H, causing it to cleave a non-target mRNA at a complementarity site (Tidd 1996). This, of course, could be counterproductive and may be used as an argument against the use of technology for crop improvement. Nevertheless, to ally the fear of off-target cleavages, the specificity of antisense action for gene silencing can be improved through some analogue modification of the backbone molecules (Fisher et al. 2002).

Outlook

There are achievements and progress recorded with the use of antisense RNA technology in crop improvement. Although, the implicit understanding of the mechanisms of antisense RNA technology is still in progress. Therefore, more antisense molecules would be subjected to scientific investigations in the future to discover the effective intercellular delivery method (s) of the ASO into the cell for the specificity of hybridization with the target mRNA. A clearer understanding of the fate of dsRNA molecules within the cytosol will enhance the utility of small RNA molecules in gene silencing. This will include a more vivid understanding of the mechanism guiding the endonucleolytic cleavage of hairpin and the precursor transcripts by RNase III-like Dicer ribonuclease and direct the cleavage of target transcripts by Argonaute RNase H-like proteins and RISC in a sequence-specific manner. In this case, more work and intensive studies are still required if the potential of the technology should be fully utilized in crop improvement to generate crop cultivars with improved agronomic traits leading to increasing yield and nutritional value towards sustainable agriculture and global food security.

Antisense RNA technology offers a promising way to decrease the expression of a particular gene, resulting in plants with modified traits. This technology is applicable in crop improvement in the areas of fruit ripening, enhanced shelf life, bacterial resistance, reduction of lignin content, etc. for man’s benefit. However, to fully utilize the technology safely, transgenic strategies using asRNA or other small RNA molecules should be designed only after a comprehensive understanding of the mechanism of regulation. This will minimize undesirable trade-offs in transgenic and reduce the controversies and ethical issues associated with the technology.

Funding: No funding was received for this work.

Declaration of Potential Conflicts of interest: The authors have no conflict of interest to declare whatsoever.

Data Availability Statement: No Data associated in the manuscript.

Ethical approval: This article does not contain any studies with animals performed by any of the authors.

Author Contribution

DAA and JAL developed the manuscript.

Aida R, Kishimoto S, Tanaka Y, Shibata M (2000) Modification of flower color in torenia (Torenia fournieri Lind.) by genetic transformation. Plant Sci 153(1):33–42. https://doi.org/10.1016/S0168-9452(99)00239-3
Animasaun DA, Azeez MA, Adubi AO, Durodola FA, Morakinyo JA (2020) Trends in genetically modified crops development in Nigeria: Issues and challenges. In: Andersen V (ed) Genetically modified and irradiated food controversial issues: Facts versus perceptions. Elsevier, Academic Press, London, pp 131–150. https://www.elsevier.com/books/genetically-modified-and-irradiated-food/andersen/978-0-12-817240-7
Aregawi K, Shen J, Pierroz G, Sharma MK, Dahlberg J, Owiti J, Lemaux PG (2022) Morphogene-assisted transformation of Sorghum bicolor allows more efficient genome editing. Plant Biotechnol J 20(4):748–760. 10.1111/pbi.13754
Atwood D, Paisley-Jones C (2017) Pesticides industry sales and usage 2008–2012 Estimates. U.S. Environmental Protection Agency Washington DC. USA. https://www.epa.gov/sites/default/files/2017-01/documents/pesticides-industry-sales-usage-2016_0.pdf
Auer C, Frederick R (2009) Crop improvement using small RNAs: Applications and predictive ecological risk assessments. Trends in Biotechnol 27(11):644–651. 10.1016/j.tibtech.2009.08.005
Bachem CWB, Speckmann GJ, van der Linde PCG, Verheggen FTM, Hunt MD, Steffens JC, Zabeau M (1994) Antisense expression of polyphenol oxidase genes inhibits enzymatic browning in potato tubers. Bio/Technol 12:1101–1105
Barry P (2007) Genome 2.0: Mountains of new data are challenging old views. Sci News 172(10):154. https://www.sciencenews.org/article/genome-20
Bird CR, Ray JA (1991) Manipulation of plant gene expression by antisense RNA. Biotechnol Genet Eng Rev 9(1):207–227. https://www.nottingham.ac.uk/ncmh/documents/bger/volume-9/bger9-6.pdf
Boland CR (2017) Non-coding RNA: it’s not junk. Dig Dis Sci 62(5):1107–1109. https://doi.org/10.1007/s10620-017-4506-1
Cagliari D, Naymã PD, Diogo MG, Ericmar AS, Guy S, Moisés JZ (2019) Management of pest insects and plant diseases by non-transformative RNAi. Front Plant Sci 10:1–14. https://doi.org/10.3389/fpls.2019.01319
Campbell TN (2005) Choy FYM, RNA interference: past, present and future. Curr Issues Mol Biol 7:1–6
Che P, Wu E, Simon MK, Anandm A, Lowe K, Gao H, Sigmund AL, Yang M, Albertsen MC, Gordon-Kamm W, Jones TJ (2022) Wuschel2 enables highly efficient CRISPR/Cas-targeted genome editing during rapid de novo shoot regeneration in sorghum. Comm Biol 5:344. 10.1038/s42003-022-03308-w
Clancy S, Brown W (2008) Translation: DNA to mRNA to protein. Nat Educ 1(1):101
Coleman CE, Larkins BA (1999) The prolamins of maize. In: Shewry PR, Casey R (eds) Seed Proteins. Kluwer Academic Press, Dordrecht, The Netherlands, pp 109–139
Crooke S (2004) Antisense strategies. Curr Mol Med 4(5):465–487. 10.2174/1566524043360375
Dash SK, Sushil KM, Malik HN (2015) RNA Interference a fine tuner of gene regulation: A review. Int J Biotechnol Mol Biol Res 6(5):35–39. 10.5897/IJBMBR2014.0213
David PC, Nanette JP (2016) RNA-based technologies in biotechnology. Second edition. Academic Press Cell, CA, USA. Pp. 397–424
Day AG, Bejarano ER, Buck KW, Burrell M, Lichtenstein CP (1991) Expression of an antisense viral gene in transgenic tobacco confers resistance to the DNA virus tomato golden mosaic virus. Proc Nat Aca Sci 88:6721–6725. 10.1073/pnas.88.15.6721
Dean NM, Bennett FC (2003) Antisense oligonucleotide-based therapeutics for cancer. Oncogene 22:9087–9096. https://doi.org/10.1038/sj.onc.1207231
Di Fusco D, Vincenzo D, Irene M, Michele MF, Barbara R, Giovanni M (2019) Antisense oligonucleotide: Basic concepts and therapeutic application in inflammatory bowel disease. Front Pharmacol, 10: Article 305. doi: 10.3389/fphar.2019.00305
Dodo HW, Konan KN, Chen FC, Egnin M, Viquez OM (2008) Alleviating peanut allergy using genetic engineering: the silencing of the immunodominant allergen Ara h 2 leads to its significant reduction and a decrease in peanut allergenicity. Plant Biotechnol J 6(2):135–145. 10.1111/j.1467-7652.2007.00292.x
Dunoyer P, Himber C, Voinnet O (2006) Induction, suppression and requirement of RNA silencing pathways in virulent Agrobacterium tumefaciens infections. Nat Genet 38:258–263. 10.1038/ng1722
El-Sappah AH, Yan K, Huang Q, Islam MM, Li Q, Wang Y, Khan MS, Zhao X, Mir RR, Li J, El-Tarabily KA, Abbas M (2021) Comprehensive mechanism of gene silencing and its role in plant growth and development. Front in Plant Sci 12:4700–5249. https://doi.org/10.3389/fpls.2021.705249
Escobar MA, Civerolo EL, Summerfelt KR, Dandekar AM (2001) RNAi-mediated oncogene silencing confers resistance to crown gall tumorigenesis. Proc Nat Aca Sci 98:13437–13442. https://doi.org/10.1073/pnas.24127689
Eric M, Cambridge MA (2018) Antisense part ii: Mechanisms of action. www.cureffi.org, Accessed date 27/02/2023
Esen A (1987) A proposed nomenclature for the alcohol-soluble proteins (zeins) of maize (Zea mays L). J Cereal Sci 5:117–128. https://doi.org/10.1016/S0733-5210(87)80015-2
Ferry N, Edwards MG, Gatehouse J, Capell T, Christou P, Gatehouse AMR (2006) Transgenic plants for insect pest control a forward looking scientific perspective. Transgenic Res 15:13–19. 10.1007/s11248-005-4803-x
Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806–811. 10.1038/35888
Fisher AA, Ye D, Sergueev DS, Fisher MH, Shaw BR, Juliano RL (2002) Evaluating the specificity of antisense oligonucleotide conjugates a DNA array analysis. J Biol Chem 277(25):22980–22984. https://doi.org/10.1074/jbc. M20334720
Frizzi A, Caldo RA, Morrell JA, Wang M, Lutfiyya L, Brown WE, Malva TM, Huang S (2010) Compositional and transcriptional analyses of reduced zein kernels derived from the opaque2 mutation and RNAi suppression. Plant Mol Biol 73(4–5):569–585. 10.1007/s11103-010-9644-1
Gelvin SB (2003) Agrobacterium-mediated plant transformation: the biology behind the “gene-jockeying” tool. Microbiol Mol Biol Rev 67:16–37. 10.1128/MMBR.67.1.16–37.2003
Godfray HCJ, Beddington JR, Crute IR, Haddad L, Lawrence D, Muir JZ, Pretty J, Robinson S, Thomas S, Toulmin C (2010) Food security: the challenge of feeding 9 billion people. Science 327:812–818. 10.1126/science.1185383
Godfrey C, Desviat LR, Smedsrød B, Piétri-Rouxel F, Denti MA, Disterer P, Lorain S, Nogales-Gadea G, Sardone V, Anwar R, El Andaloussi S, Lehto T, Khoo B, Brolin C, van Roon-Mom WM, Goyenvalle A, Aartsma-Rus A, Arechavala-Gomeza V (2017) Delivery is key: lessons learnt from developing splice-switching antisense therapies. EMBO Mol Med 9(5):545–557. https://doi.org/10.15252/emmm.201607199
Gordon KHJ, Waterhouse PM (2007) RNAi for insect-proof plants. Nat Biotechnol 25:1231–1232. 10.1038/nbt1107-1231
Gupta S, Singh RP, Rabadia N, Patel G, Panchal H (2011) Antisense technology. Int J Pharm Sci Rev Res 9:38–40 www.globalresearchonline.net
Gupta A, Pal RK, Rajam MV (2013) Delayed ripening and improved food processing quality in tomato by RNAi-mediated silencing of three homologs of 1-aminopropane-1-carboxylate synthase gene. J Plant Physiol 170(11):987–995. 10.1016/j.jplph.2013.02.003
Hamada H, Liu Y, Imai R (2018) Biolistic-delivery-based transient CRISPR/Cas9 expression enables in Planta genome editing in wheat. Sci Rep 8:14422. https://doi.org/10.1038/s41598-018-32714-6
Jens K (2003) Antisense technologies: Improvement through novel chemical modifications. Eur J Biochem 270:1628–1644. 10.1046/j.1432-1033.2003.03555.x
Ji T, Lu A, Wu K (2017) Antisense RNA insert design for plasmid construction to knockdown target gene expression. Undergrad Methods Paper 1:7–15. https://jemi.microbiology.ubc.ca/sites/default/files/Ji%20et%20al%20JEMI-methods%20Vol%201%20pg%207-15.pdf
Kelly WG, Aramayo R (2007) Meiotic silencing and the epigenetics of sex. Chromato Res 15:633–651. 10.1007/s10577-007-1143-0
Kim J, Chang C, Tucker ML (2015) To grow old: regulatory role of ethylene and jasmonic acid in senescence. Front Plant Sci 6. : Article 20. https://doi.org/10.3389/fpls.2015.00020
Kumar A (2010) Gene: Expression and regulation. In: Recent advances in Life Sciences, 1st Edition. IK International Publisher, New Delhi, India. Pp. 353–375
Kumar S, Bejiga G, Ahmed S, Nakkoul H, Sarker A (2011) Genetic improvement of grass pea for low neurotoxin (β-ODAP) content. Food Chem Toxicolo 49(3):589–600. https://doi.org/10.1016/j. fct.2010.06.051
Kumar P, Kamle M, Pandey A (2012) RNAi: New era of functional genomics for crop improvement. Front Recent Dev Plant Sci 1:24. https://doi.org/10.2174/978160805403911201010024
Lackner DH, Bahler J (2008) Translational control of gene expression: From Transcripts to transcriptomes. Int Rev Cell Mol Biol 271:199–251. 10.1016/S1937-6448(08)01205-7
Le NT, Wang M (2011) The role of RNA silencing in plant stress responses, Omics and Plant Abiotic Stress Tol 1: 151. Bentham Science Publisher. Sharjah, U.A.E. https://doi.org/10.2174/978160805058111101010151
Lemmon ZH, Reem NT, Dalrymple J, Soyk S, Swartwood KE, Rodriguez-Leal D, Van Eck J, Lippman ZB (2018) Rapid improvement of domestication traits in an orphan crop by genome editing. Nat Plants 4(10):766–770. https://doi.org/10.1038/s41477-018-0259-x
Luckanagul JA, Lee LA, You S, Yang X, Wang Q (2014) Plant virus incorporated hydrogels as scaffolds for tissue engineering possess low immunogenicity in vivo. J Biomedic Mat Res: Part A 103(3):887–895. 10.1002/jbm.a.35227
Matzke M, Matzk A, Pruss G, Vance V (2001) RNA-based silencing strategies in plants. Curr Opin Genet Dev 11:221–227 S0959- 437X(00)00183-0
Matzke MA, Birchler JA (2005) RNAi-mediated pathways in the nucleus. Nature Rev Genet 2005: 6(1):24–35
Miflin B (2000) Crop improvement in the 21st century. J Exp Bot 51(342):1–8. https://doi.org/10.1093/jexbot/51.342.1
Mo Y, Wan R, Zhang Q (2012) Application of reverse transcription-PCR and real-time PCR in nanotoxicity research methods. Mol Biol 926:99–112. 10.1007/978-1-62703-002-1_7
Mol JNM, Vander KAR, Van-Tunen RAJ, Van-Blokland P, De L, Stuitje AR (1990) Regulation of plant gene expression by antisense RNA. Fed Eur Biochem Soc Lett 268(2):427–430. https://doi.org/10.1016/0014-5793(90)81298-3
Mu X, Greenwald E, Ahmad S, Hur S (2018) An origin of the immunogenicity of in vitro transcribed RNA. Nucleic Acids Res 46:5239–5249
Nakatsuka T, Mishiba KI, Kubota A, Abe Y, Yamamura S, Nakamura N, Tanaka Y, Nishihara M (2010) Genetic engineering of novel flower colour by suppression of anthocyanin modification genes in gentian. J Plant Physiol 167:231–237. https://doi.org/10.1016/j.jplph.2009.08.007
Nguyen T, Shanklin J (2008) Altering Arabidopsis oilseed composition by a combined antisense-hairpin RNAi gene suppression approach. J Ameri Oil Chemists' Soc 2008: 86:41–49
Nishihara M, Nakatsuka T, Yamamura S (2005) Flavonoid components and flower color change in transgenic tobacco plants by suppression of chalcone isomerase gene. Fed Eur Biochem Soc Lett 579:6074–6078. https://doi.org/10.1016/j.febslet.2005.09.073
Nizampatnam NR, Kumar VD (2011) Intron hairpin and transitive RNAi mediated silencing of orfH522 transcripts restores male fertility in transgenic male sterile tobacco plants expressing orfH522. Plant Mol Biol 76(6):557–573. https://doi.org/10.1007/s11103-011-9789-6
Oberemok VV, Laikova KV, Gal’chinsky NV, Useinov RZ, Novikov IA, Temirova ZZ, Shumskykh MN, Krasnodubets AM, Repetskaya AI, Dyadichev VV, Iryna I, Fomochkina II, Bessalova EY, Makalish TP, Yuri I, Gninenko YI, Kubyshkin AV (2019) DNA insecticide developed from the Lymantria dispar 5.8S ribosomal RNA gene provides novel biotechnology for plant Protection. Sci Rep 9:619
Oeller PW, Lu MW, Taylor LP (1991) Reversible inhibition of tomato fruit senescence by antisense RNA. Science 254(5030):437–439. 10.1126/science.1925603
Pandey AK, Madhu P, Bhat BV (2019) Down-regulation of CYP79A1 gene through antisense approach reduced the cyanogenic glycoside dhurrin in Sorghum bicolor (L.) Moench to improve fodder quality. Front Nutri 6:122. https://doi.org/10.3389/fnut.2019.00122
Pare JM, Hobman TC (2007) Dicer: structure, function and role in RNAdependent gene-silencing pathways. In: Polania J, MacCabe AP (eds) Industrial Enzymes. Springer, Berlin, pp 421–438
Powell PA, Stark DM, Sanders PR, Beachy RN (1989) Protection against tobacco mosaic virus in transgenic plants that express tobacco mosaic virus antisense RNA. Proc Natl Aca Sci USA 86(18):6949–6952. 10.1073/pnas.86.18.6949
Pradeep K, Satya VK, Selvapriya M, Vijayasamundeeswari A, Ladhalakshmi D, Paranidharan V, Rabindran R, Samiyappan R, Balasubramanian P, Velazhahan R (2012) Engineering resistance against Tobacco streak virus (TSV) in sunflower and tobacco using RNA interference. Biol Plant 56:735–741. https://doi.org/10.1007/s10535-012-0111-5
Rajam MV (2020) RNA silencing technology: A boon for crop improvement. J Biosci 45(1):1–5. 10.1007/s12038-020-00082-x
Rajput M, Choudhary K, Kumar M, Vivekanand V, Chawade A, Ortiz R, Pareek N (2021) RNA interference and CRISPR/Cas gene editing for crop improvement: Paradigm shift towards sustainable agriculture. Plants, 10(9): 1914. https://doi.org/10.3390/plants10091914
Reese CB (2005) Oligo-and poly-nucleotides: 50 years of chemical synthesis. Org Biomole Chem 3(21):3851–3868. https://pubs.rsc.org/en/content/articlepdf/2005/ob/b510458k
Riley KJ, Yario TA, Steitz A (2012) Association of argonaute proteins and microRNAs can occur after cell lysis. RNA 18:1581–1585. 10.1261/rna.034934.112
Rusk N (2015) Understanding noncoding RNAs. Nat Methods 12(1):35. https://doi.org/10.1038/nmeth.3235
Salehuzzaman SNIM, Jacobsen E, Visser RGF (1993) Isolation and characterization of a cDNA encoding granule-bound starch synthase in cassava (Manihot esculenta Crantz) and its antisense expression in potato. Plant Mol Biol 23(5):947–962. https://doi.org/10.1007/Bf00021811
Sandhu APS, Abdelnoor RV, Mackenzie SA (2007) Transgenic induction of mitochondrial rearrangements for cytoplasmic male sterility in crop plants. Proc Nat Aca Sci USA 104(6):1766–1770. https://doi.org/10.1073/pnas.0609344104
Saurabh S, Vidyarthi AS, Prasad D (2014) RNA interference: Concept to reality in crop improvement. Planta 239(3):543–564. 10.1007/s00425-013-2019-5
Scherer LJ, Rossi JJ (2003) Approaches for the sequence specific knockdown of mRNA. Nat Biotechnol 21:1457–1465. 10.1038/nbt915
Schwind N, Zwiebel M, Itaya A, Ding B, Wang M, Krczal G, Wassenegger M (2009) RNAi-mediated resistance to potato spindle tuber viroid in transgenic tomato expressing a viroid hairpin RNA construct. Mol Plant Pathol 10:459–469. https://doi.org/10.1111/j.1364-3703.2009.00546.x
Seitz C, Vitten M, Steinbach P, Hart S, Hirsche J, Rathje W, Treutter D, Forkmann G (2007) Redirection of anthocyanin synthesis in Osteospermum hybrida by a two-enzyme manipulation strategy. Phytochem 68(6):824–833. https://doi.org/10.1016/j.phytochem.2006.12.012
Sestili F, Janni M, Doherty A, Botticella E, D’Ovidio R, Masci S, Jones HD, Lafiandra D (2010) Increasing the amylose content of durum wheat through silencing of the SBEIIa genes. BMC Plant Biol 10(1):144. https://doi.org/10.1186/1471-2229-10-144
Simón-Mateo C, García JA (2011) Antiviral strategies in plants based on RNA silencing. Biochim Biophys Acta 1809:722–731. https://doi.org/10.1016/j.bbagrm.2011.05.011
Singh RK, Krishnamachari A, Sharaff M (2020) Challenges of small RNA technology. In: Praveen Guleria, Vineet Kumar (eds), Plant Small RNA, Elsevier pp. 545–565
Sivaraman I, Arumugam N, Sodhi Y, Gupta V, Mukhopadhyay A, Pradhan A et al (2004) Development of high oleic and low linoleic acid transgenics in a zero erucic acid Brassica juncea L. (Indian mustard) line by antisense suppression of the FAD2 gene. Mol Breed 13:365–375
Siritungam D, Sayre R (2003) Generation of cyanogen-free transgenic cassava. Planta 217:367–373. 10.1007/s00425-003-1005-8
Sohrab SS, Kamal MA, Ilah A, Husen A, Bhattacharya PS, Rana D (2016) Development of Cotton leaf curl virus resistant transgenic cotton using antisense ßC1 gene. Saudi J Biolo Sci 23(3):358–362
Stephanie H (2020) What is antisense technology. Azo Life Sci. 1: 1–6. i>www.azolifescience.com
Tanmoy A, Alum M, Islam M, Farzana T, Khan H (2014) Jute (corchorus olitorius var. O-72) stem lignin: variation in content with age. Bangl J Bot 43(3):309–314. 10.3329/bjb.v43i3.21603
Tidd DM (1996) Specificity of antisense oligonucleotides. Perspect Drug Disc Des 4(1):51–60. https://doi.org/10.1007/BF02172108
Tilahun T, Bezie Y, Kerisew B, Taye M (2021) The application of antisense technology for crop improvement: A review. Cogent Food and Agric 7:157–191. https://doi.org/10.1080/23311932.2021.1910157
Tiwari M, Sharma D, Trivedi PK (2014) Artificial microRNA mediated gene silencing in plants: progress and perspectives. Plant Mol Biol 86(1–2):1–18. https://doi.org/10.1007/s11103-014-0224-7
Tuck GA, Seymour GB, Bundick Y, Robertson D, Hall L, Smith CJS, Grierson D, Bird CR, Schuch W (2016) Use of antisense RNA technology to study pectin degradation in tomato fruit. New Z J Crop Hortic Sci 20(2):119–124. 10.1080/01140671.1992.10421904
Tram K, Wang X, Yan H (2007) Facile synthesis of oligonucleotide phosphoroselenoates. Org Lett 9(24):5103–5106. https://doi.org/10.1021/ol702305v
van der Krol AR, Mol JN, Stuitje AR (1988) Modulation of eukaryotic gene expression by complementary RNA or DNA sequences. Biotechniques 6(10):958–976
van der Meer IM, Stam ME, van Tunen AJ et al (1992) Antisense inhibition of flavonoid biosynthesis in petunia anthers results in male sterility. Plant Cell 4(3):253–262. https://doi.org/10.1105/tpc.4.3.253
Wakimoto BT (1998) Beyond the nucleosome: epigenetic aspects of position-effect variegation in Drosophila. Cell 93:321–324. https://doi.org/10.1016/S0092-8674(00)81159-9
Walder RY, Walder JA (1988) Role of RNase H in hybrid-arrested translation by antisense oligonucleotides. Proc Nat Aca Sci 85:5011–5015. https://doi.org/10.1073/pnas.85.14.5011
Wang N, Ryan L, Sardesai N, Wu E, Lenderts B, Lowe K, Che P, Anand A, Worden A, van Dyk D, Barone P, Svitashev S, Jones T, Gordon-Kamm W (2023) Leaf transformation for efficient random integration and targeted genome modification in maize and sorghum. Nat Plants 9:255–270. https://doi.org/10.1038/s41477-022-01338-0
Waterhouse PM, Graham HW, Wang M (1998) Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proc Nat Aca Sci 95:13959–13964. 10.1073/pnas.95.23.13959
Watson JD, Crick FHC (1953) A structure for deoxyribose nucleic acid. Nature 171(4356):737–738. https://doi.org/10.1038/171737a0
Wilson RC, Doudna JA (2013) Molecular mechanisms of RNA interference. Annu Rev Biophys 42:217–239. 10.1146/annurev-biophys-083012-130404
Woodward F (2022) A Brief Note on Transcriptional and Post-Transcriptional Gene Silencing. J Genet Synd Gene Therap 13:372. 10.35248/ 2157-7412.22.13.372
Vaucheret H, Béclin C, Fagard M (2001) Post-transcriptional gene silencing in plants. J Cell Sci 114:3083–3091. https://doi.org/10.1242/jcs.114.17.3083
Xie D, Dai Z, Sun J, Su J (2020) Full-length trans sequencing provides insight into lignan and lignin metabolism relation. Biotechnol Biotechnolog Equip 34:1122–1133. 10.1080/13102818.2020.1825122
Xiong A, Yao Q, Peng R, Li X, Han P, Fan H (2005) Different effects on ACC oxidase gene silencing triggered by RNA interference in transgenic tomato. Plant Cell Rep 23:639–646. 10.1007/s00299-004-0887-7
Xu H, Wei Y, Zhu Y, Lian L, Xie H, Cai Q et al (2015) Antisense suppression of LOX3 gene expression in rice endosperm enhances seed longevity. Plant Biotechnol J 2015:13(4):526 – 39
Xu J, Zhang J, Zhang W (2018) Antisense RNA: the new favorite in genetic research. J Zhejiang University-Science B (Biomed Biotechnol 19(10):739–749. 10.1631/jzus.B1700594
Yan X, Corbin K, Burton RA, Tan DKY (2020) Agave: A promising feedstock for biofuels in the water-energy-food-environment (WEFE) nexus. J Clean Prod 261. https://doi.org/10.1016/j.jclepro.2020.121283
Zamecnik PC, Stephenson ML (1978) Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide. Proc Nat Aca Sci 75:280–284. 10.1073/pnas.75.1.280
Zaramella S, Yeheskiely E, Stromberg R (2004) A Method for Solid-Phase Synthesis of Oligonucleotide 5‘-Peptide-Conjugates Using Acid-Labile α-Amino Protections. J Amer Chem Soc 126(43):14029–14035. https://doi.org/10.1021/ja046945o
Zebec Z, Zink IA, Kerou M, SchleperC (2016) Efficient CRISPR-mediated post-transcriptional gene silencing in a hyperthermophilic archaeon using multiplexed crRNA expression. Genes Geno Genet 36:3161–3168. 10.1534/g3.116.032482
Zhang L, Yang XD, Zhang YY, Yang J, Qi GX, Guo DQ et al (2014) Changes in oleic Acid content of transgenic soybeans by antisense RNA mediated posttranscriptional gene silencing. Int J Geno 2014:921–950
Zhang Z, Yang J, Wu Y (2015) Transcriptional regulation of zein gene expression in maize through the additive and synergistic action of opaque2, prolamine-box binding factor, and O2 heterodimerizing proteins. Plant Cell 27:1162–1172. 10.1105/tpc.15.00035

Download PDF

Journal Publication

published 27 Sep, 2023

Read the published version in Molecular Biology Reports →

Editorial decision: Minor Revisions Needed
09 Aug, 2023
Reviewers agreed at journal
27 May, 2023
Reviewers invited by journal
26 May, 2023
Editor assigned by journal
25 May, 2023
First submitted to journal
22 May, 2023

You are reading this latest preprint version

Antisense RNA (asRNA) Technology: The Concept and Applications in Crop Improvement and Sustainable Agriculture

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Gene silencing

Antisense RNA

Synthesis of antisense oligonucleotides

Principles and mechanism of antisense RNA technology

Methods of delivery of the asRNA into the cell

Applications of antisense RNA technology in crop improvement

Virus resistance

Developing bacterial-resistant crops

Pest resistance

Reduction of linamarin in cassava

Reduction of cyanogenic glycoside in sorghum

Reduction of lignin content in jute

Enhancement of nutritional value and quality

Inducement or restoration of male sterility

Challenges

Outlook

Conclusion

Declarations

References

Status:

Journal Publication

Version 1