The foremost concern of SMGs in engineered crops is socio-ethical issues and transgene expression. Even several copies of SMGs may result in the silencing of the essential genes of plants and affect plant metabolism (Rosellini, 2012). The batteries of methods have been developed to make marker-free transgenic crops, including rice, as discussed below.

Recently, the most widely used genome editing tool known as CRISPR/Cas9 has also been brought into use to remove or avoid SMG in transgenic rice. Using the CRISPR/Cas9 tool, site-specific DSB is induced at the target site, followed by a repair mechanism either through homology-directed repair (HDR) or non-homologous end joining (NHEJ). Among both, the natural occurrence of HDR is rare and thus requires a donor template to repair DSB (Zafar et al., 2020). The delivery of donor templates is quite challenging due to the difficulties of its delivery and short-time stability in the cell. Therefore, recent efforts have aimed to increase HDR efficiencies, such as geminivirus-based donor template delivery (Wang et al., 2017) and Cas9-VirD2 chimeric protein (Ali et al., 2020). HDR-based SMG excision and marker-free gene insertion have been achieved (discussed next). It is imperative to mention that CRISPR is a more precise, efficient, and less time-consuming technology. Traditional methods, like co-transformation (using two independent T-DNA plasmids), transposon and recombinase systems (which leave cryptic sequences in the host genome) need a large screening population to segregate SMG. In contrast, the CRISPR/Cas9 based SMG-free approach utilizing HDR does not leave any foreign sequences in the genome. Moreover, RNP-based genome editing is considered DNA-free, and thus does not incorporate plasmid DNA sequences in the genome. It has now become possible to get rid of selectable markers as well as transgene cassettes that persisted in the plant genomes. The utilization of CRISPR/Cas9 as an SMG-free tool has been reported in the last few years and is in infancy. However, much is expected from this technology in terms of making SMG-free rice in the future. To date, only a few studies have reported the successful use of CRISPR/Cas9 as an SMG-free technique in rice, as discussed below.

The RNP complex is constituted by nuclease and guide RNA is DNA- and SMG-free approach. Earlier, RNP-based edited rice plants have been generated for grain size and grain weight (Toda et al., 2019; Banakar et al., 2020). The fragrance is considered one of the essential grain quality traits in rice as it determines the market price. The aroma in rice is associated with an increased amount of 2-acetyl-1-pyrroline (2AP) controlled by the betaine aldehyde dehydrogenase2 (badh2) gene (Buttery et al., 1983). The sequence alignment of the OsBADH2 gene among non-fragrant and fragrant lines revealed few mutations i.e., 8-bp deletion and three SNPs in exon 7, 7-bp deletion in exon 2, and 803-bp (intronic) deletions between exon 4 and 5 (Shan et al., 2015). These mutations introduce a premature stop codon upstream of key coding regions, making this gene non-functional (badh2) (Hashemi et al., 2013; Shan et al., 2015). A few attempts have been made to introduce aroma in non-aromatic rice through RNAi (Niu et al., 2008) and genome-editing approaches. Recently, Ashokkumar et al. (2020) successfully created novel alleles in rice variety ASD16 by knocking out the OsBADH2 gene through a vector-based CRISPR approach.

In our laboratory, we attempted the editing of the OsBADH2 gene in non-aromatic rice. Basmati rice belongs to aromatic rice that has a pleasant and exquisite aroma with a low yield. However, elite cultivar PR114 lacks aroma in contrast to basmati rice. Its average yield is 6.9 tons per hectare, whereas, Basmati varieties have an average yield of 4.0 tons per hectare. The introduction of aroma in PR114 without disturbing its original genetic constitution will lead to premium quality aromatic high-yielding rice. It would lead to a major revolution for the stakeholders. A total of 1,100 embryos were bombarded by the RNP complex coated gold particles for exon 2 and exon 7 using the protocols outlined by Banakar et al. (2020). In total, 731 embryos were germinated under in vitro conditions on MS synthetic media, and 253 plantlets were transferred to soil. Only 35 plants survived in a glasshouse (Figure 3A), screened using the MSBSP-PCR (Mutation Site-Based Specific Primers-PCR) technique (Guo et al., 2018). Seven putative edited plants were obtained through the MSBSP-PCR (Figure 3B) and were subjected to Sanger sequencing (Figure 3C). The sequences of putative edited plants were aligned against the PR114 reference sequence using Clustal Omega software, which revealed the addition of a nucleotide “A” at 4-bp upstream of PAM sequence in the target site of the edited plant # 11–4 ( Plant no. 11, tiller no. 4; Figure 3D). The alignment of the amino acid sequence of PR114 (Figure 3E) and plant 11–4 using the Expasy online tool showed the frameshift mutation in exon 7 (Figure 3F). The confirmed T₀ plant progeny will be raised and screened through molecular and biochemical analysis. To the best of out knowledge, this is the first report on RNP-based OsBADH2 gene editing.

The presence of SMGs, especially hpt, npt II, and Bar genes in transgenic rice is one of the major hurdles in their regulatory approval. The reasons behind their strict regulations are; the spread of their resistance in natural flora and fauna, and unintended changes in plant transcriptome and metabolome (pleiotropic effect) (Chong-Pérez and Angenon, 2013). Newly developed food that is genetically modified (GM) for a particular trait has to go through rigorous testing at molecular, biochemical, and metabolic levels for food and feed safety (including toxicity, allergenicity, and anti-nutrient). This process also makes sure the claims of substantial equivalence to non-GM wild type phenotypes are valid and that the genetically modified food is safe for environmental release (Giraldo et al., 2019). Regulatory concerns related to the presence of SMGs and the importance of their withdrawal from gene cassettes needed for further approval are apparent in a few examples of GM rice events produced in the past. The first best example of transgenic rice is “Xianyou 63”, approved for release by China through co-transformation of two separate plasmids harboring cry1Ab/Ac and hpt selectable marker, respectively. The events developed were passed through the regulatory regime, and molecular characterization revealed the insertion of truncated hpt gene fragments (Lu, 2010). Another case is Golden rice 1 (GR1), harboring gene cassette for beta-carotene and hpt as a selectable marker. Event GR1 was unacceptable due to public concerns about the hpt marker gene. Thus, another event GR2, with a higher accumulation of beta-carotene than GR1, was produced by Syngenta using the pmi gene (Paine et al., 2005).

From its early development, the Golden rice trait (from GR2E event with single gene copy) has been successfully introgressed into elite rice cultivars viz. R64, PSBRc82, and BR29 using backcross breeding (MallikarjunaSwamy et al., 2021). After facing all the regulatory parameters, the GR2E event has been approved for consumption in different continental parts, including Australia, Canada, New Zealand, the Philippines, and the United States (https://www.goldenrice.org/). The regulation of newly developed GMOs comes under three categories. 1) process-based (for example, Europe) where the overall process or technique used to make GMO is regulated, 2) product-based (for example, the United States) where the only final product is regulated, and 3) both at the process as well as product-level regulation (for example, India). The major opponents of Golden rice are the European Union (EU), where regulation is applied to food and feed products and is a process-based regulatory scheme (https://www.ncbi.nlm.nih.gov/books/NBK424533/). Even genome-edited crops using CRISPR/Cas9 were also included in the definition of GMO as per the European Court of Justice (ECJ) in 2018 (Turnbull et al., 2021). In contrast, North America and especially the United States do not have any specific federal laws for the process regulation through which GMOs are produced. The newly developed GM products are directed to specialized regulatory bodies to assess the health, safety, and environmental laws, which are the same as those used for conventional products. In Africa, the two main approaches for seed development include biotechnology and conventional, which contribute to food and nutritional security. The former is regulated under the Biosafety act and later through the Seed act and is often accompanied by National Performance Trials (NPTs) (Akinbo et al., 2021) to ensure harmony in decision making.

Research that aims to create SMGs-free transgenic crops has always encouraged plant molecular biologists to adopt new ways to remove selectable markers from the GM plant background. The most widely used method is co-transformation. However, it is laborious to screen a segregating population for SMG-free plants and even could not be possible in vegetatively propagated crops (Breyer et al., 2014). Alternative to the traditional methods, CRISPR/Cas9-based genetic manipulations enable the development of SMG-free crops easily and precisely. The CRISPR/Cas9 method to make SMG-free rice is at the initial stage and few attempts have been made to improve the technique. The CRISPR-based npt II marker degradation in transgenic tobacco has been reported by Rezaei et al. (2021) and programmed self-elimination in rice by He et al. (2018). These techniques could pave the way to making SMG-free engineered plants in the future.

There are a few limitations of the RNP-mediated genome-editing through CRISPR: 1) the low transformation efficiency of RNP; 2) the difficulty of screening plants; and 3) using embryos as an explant shows chimerism in the T₀ stage. A novel strategy has been proposed to overcome these limitations. Studies in rice have reported that over ten distinct plasmids could be delivered together into the plant genome by particle bombardment (Chen et al., 1998). The transformation of two plasmids using a biolistic gene gun exhibited a higher frequency (85%) in contrast to a single plasmid (Hilliou et al., 1999). The co-delivery of RNP and plasmid with selectable markers is a highly beneficial technique (Banakar et al., 2019). The RNP complex can edit the target gene without the integration of CRISPR elements into the genome and reduces the number of off-targets due to transient presence. The selectable marker in the plasmid facilitates the easy selection of transformed plants. This technique combines the benefits of the targeted mutation of RNP-mediated transformation and the easy selection process of a selectable marker in a plasmid. He et al. (2018) demonstrated the technique, TKC (Trangene Killer CRISPR), for the elimination of plasmids from the mutated plant using the suicidal gene (BARNASE) under the control of REG2 promoter (expressed during the early embryo development stage).

A combination of two approaches, the co-delivery of the RNP complex along with a gene cassette consisting of a suicidal gene and antibiotic-selectable markers (hpt, npt II, etc.) has been proposed as a new method of genome editing that is DNA and marker-free (Figure 4). In this approach, three scenarios are formed: 1) RNP transformed cells; 2) cells transformed with both RNP and cassette; and 3) cells transformed with only cassette. Transformed cells with RNP-cassette and cassette only would survive during the first screening step using selective media, while transformed plants with RNP only would be lost. In the second round of selection by MSBSP-PCR, the T₀ plants with cassette only would be eliminated. The plants with both RNP and cassette would be selected and advance to the next generation. When these screened plants reach the seed setting stage, embryos with cassette would be killed as per the Programmed Self elimination effect, whereas plants with mutated target site lacking cassette would survive. Hence, seeds obtained from the T₀ generation would be DNA and marker-free edited plants. We hypothesize that a straightforward and novel approach to making marker-free engineered crops for food security will support developing countries in introducing the product, thus contributing to the prologue of these products all over the world.