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Selectable Markers to Marker‑Free Selection in Rice

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AnnuMinhas

The document reviews the advancements in rice biotechnology, focusing on selectable marker genes (SMGs) used for plant transformation in achieving stress tolerance. It outlines various selection strategies to eliminate foreign DNA elements post-transformation, emphasizing the importance of systems like CRISPR/Cas9 for efficient marker-free selection. The review analyzes the critical role of genetic engineering in improving rice varieties against abiotic and biotic stresses to enhance agricultural productivity worldwide.

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Vol.:(0123456789) 1 3 Molecular Biotechnology https://doi.org/10.1007/s12033-022-00460-w REVIEW Selectable Markers to Marker‑Free Selection in Rice Aditi Sharma1 · Ayush Chouhan1 · Tarun Bhatt1 · Anupreet Kaur1 · Anu Priya Minhas1 Received: 21 August 2021 / Accepted: 3 February 2022 © The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2022 Abstract Inadequate rice production worldwide is largely attributed to abiotic and biotic stresses, along with high sensitivity of cultivable plant germplasm. In the field of cereal biotechnology, rice engineering plays an important role in achieving tol- erance to such stresses. Plant transformation and selection play crucial role in rice engineering. This review summarized the antibiotic, herbicide and metabolic selection marker genes (SMG) employed in diverse rice engineering studies. These SMGs are no longer required after the transformation has been achieved, hence undesirable at the commercial level. This study also included several strategies employed in rice engineering to eliminate such foreign DNA elements. These include co-transformation, site-specific recombination, transposon and CRISPR base approaches. CRISPR/Cas9 being simple and efficient, is considered a crucial step toward clean gene technology. Further ease and applicability of CRISPR/Cas9 in the embryos directly can help us to modify target genes with efficient marker-free selection in minimum time. Overall, this review summarizes and analyse the recent advances that have enormous potential in rice improvement. Keywords Rice · Selection marker gene · Marker-free selection · CRISPR · Transformation · Engineering Abbreviations SMG Selectable marker gene nptII Neomycin phosphotransferase II encoding gene EPSPS 5-Enolpyruvyl shikimate 3-phosphate synthase hpt Hygromycin phosphotransferase encoding gene GM Genetically modified hph Hygromycin B phosphotransferase encoding gene T-DNA Transfer DNA FLP/FRT Flippase/FLP recombinase recognition target CRE/loxP Cre recombinase/locus of x-over, P1 CRISPR Clustered regularly interspaced short palin- dromic repeats Cas CRISPR associated protein Introduction Rice (Oryza sativa L.) is the second most cultivated cereal crop worldwide [1]. Interestingly, every second person on this planet relies on rice as the staple food. Asia alone holds the major share (above 90%) of world’s rice production and consumption. There are 24 species in the genus Oryza, only two are domesticated. Oryza sativa is an Asian rice, widely cultivated in southern and eastern Asia. The African rice, Oryza glaberrima, is native to and confined mostly to western Africa. O. sativa, O. glaberrima and their wild progenitors are diploids (2n=24) with AA genomes [2, 3]. In the past, O. sativa has been divided into two subspecies, Japonica and Indica, but recent genetic studies suggest five distinct groups of rice [4]. From 1996 to 2011, rice produc- tion rose by 180% from 257 million tons to 718 million tons owing to the green revolution. However, population explo- sion in low-income countries with 800 million people has Aditi Sharma and Ayush Chouhan have contributed equally. * Anu Priya Minhas annuminhas@gmail.com Aditi Sharma aditisharma102@gmail.com Ayush Chouhan ayushc125@gmail.com Tarun Bhatt tarunbhatt0300@gmail.com Anupreet Kaur anupreetz@yahoo.co.in 1 Department of Biotechnology, University Institute of Engineering and Technology, Sector‑25, Panjab University, Chandigarh 160014, India
Molecular Biotechnology 1 3 paralyzed us to get sufficient food for everyone. The ultimate vision for 2050 is to produce staple foods on the same piece of arable land for this increased population [5, 6]. Need of Genetic Engineering in Rice Crop In rice crop, many promising varieties have the potential to produce 10 ton per hectare yield under controlled environ- ment. However, local farmers under field conditions end up harvesting half of the actual yield reasoned to various envi- ronmental stresses [7]. Drought, salinity, submergence, cold, high temperature, and metal toxicity are examples of biotic (insect and pest) and abiotic (drought, salinity, submergence, cold, high temperature, and metal toxicity) environmental stresses. Drought stress is one of the abiotic stresses that poses a severe threat to world rice production when water is scarce. Drought stress alone has been estimated to reduce worldwide rice production by 18 million tons annually. It affects plant growth by reducing cell growth, cell elonga- tion, and cell expansion. As a result, reactive oxygen species build up, damaging the plant's antioxidant system. By regu- lating stress-induced gene and protein functions, drought stress modifies morphological, biochemical, physiological, and molecular responses. Salt concentrations in the soil is another crucial factor affecting rice plant development at all stages by causing ionic or osmotic stress [8–10]. Excess of sodium ions compete with useful ions and upset the ionic balance. Osmotic stress can influence the plant’s ability to absorb water from the soil. Reduction in cellular water potential due to the increased solute concentration affects stomatal conductance, transpiration, gaseous exchange and rate of carbon assimilation [11, 12]. Complete submer- gence also affects many upland rice cultivars critically. A moderate waterlogged environment can extend leaves and stems, resulting in excessive energy use and death of cells [13, 14]. Low temperatures also affect rice germination, seedling growth, leaf curving, shoot length, flowering and tillering [15, 16]. Further, due to the limited variety of cul- tivable rice cultivars and the labour intensive agriculture techniques, rice cultivation is seasonal. In order to develop varieties resistant to these stresses, it is essential to under- stand how plants respond to these stresses. Biggest challenge in plant biotechnology is to develop stress tolerant plants employing genetically engineered technology. Traditional crop improvement practices such as plant breeding have their own limits. At present, genetic engineering is utilized to increase the genetic pool of crop species. As a result, genes from a variety of organisms can be introduced with new or enhanced functions when exposed to certain biotic and abi- otic stresses. Being quick, accurate, and stable, genetic engi- neering is speculated as superior alternative method. How- ever, exact selection of true transgenics with high precision depends on rigorous selection procedures. Through the transfer of desirable genes, this technology allows access to an unlimited gene pool. The transformation method quiet efficient to produce useful plants with special phenotypes within a short period of time, rectify faults, and improve physiological and agronomical traits [5]. DNA introduction and selection of true transformed plant cells/tissue constitute two most demanding steps of rice transformation [17]. Rice plant transformation involves introducing foreign DNA seg- ments with a selectable marker gene (SMG) into either plant nuclear DNA or chloroplast genome [18]. Proteins encoded by SMGs confer competitive advantage on only transformed cells, making them more likely to be selected in presence of a selection agent over untransformed cells. SMGs Antibiotic/herbicide resistance genes are initially employed for successfully selecting transgenic model plants resistant to various biotic and abiotic traits. Later, the same technology has been extended to crops including rice and other cere- als successfully [19, 20] (Table1). Transformed cells show resistance to antibiotics when antibiotic resistance genes from bacterial origin are expressed under plant-specific pro- moters. Glycopeptides and aminoglycosides constitute major classes of antibiotics employed in plant research. Hygromy- cin, kanamycin, neomycin, gentamicin, geneticin (G418) and paromomycin are of aminoglycoside types whereas bleomy- cin belongs to glycopeptide family [21]. Antibiotics in the aminoglycoside family work as protein inhibitors, halting cell growth [22]. Kanamycin, gentamicin, geneticin and neomycin block protein synthesis by binding 30S subunit in ribosome whilst hygromycin occupies the ribosome binding site in the elongation factor EF-2 [23]. Plants’ sensitivity, on the other hand, varies greatly between species, genotypes, and tissues [24–27]. Antibiotic Resistant Genes as SMGs Neomycin phosphotransferase II (aphA2), expressed by EPSPS gene, from Escherichia coli transfer phosphate molecule to their respective hydroxyl groups and confers resistance to kanamycin and G418. The phosphate molecule hinders with the interactions and binding of antibiotics to ribosomes. Same has also been employed as a selection marker in rice as well. Kanamycin (150 mg/l) along with cefotaxime has been used for selecting Indica rice (Oryza sativa L.) cv. RD6 transformants [28]. Chan et al. report the transformation of Indica rice cv. Taichung Native 1 using nptII gene and select the transformed rice using 20 mg/l of kanamycin [29]. Tripathi et al. attempt to increase
Molecular Biotechnology 1 3 Table 1  List of rice transformation studies indicating their corresponding SMG, selection agents, mode of action, transformation method, plant part used and MIC of the selection agent Selection agent Mode of action Resistance gene and encoded enzyme Rice used in the study Transformation tech- nique used Plant part used MIC value of selection agent References Aminoglycosides Kanamycin, Geneticin (G418) Binds to the 30S riboso- mal subunit nptII, Neomycin phosphotransferase II enzyme (NPT) Oryza sativa L Electroporation Rice protoplast 200 mg/l Dekeyser et al. [38] (Oryza sativa L.) cv. RD6 Agrobacterium-medi- ated transformation Rice callus 150 mg/l Thodsaporn et al. [28] Indica rice cv. Taichung native 1 Agrobacterium-medi- ated transformation Rice callus 20 mg/l Chan et al. [29] Pusa Basmati-1 (IET- 10364) Agrobacterium-medi- ated transformation Rice callus 50 mg/l Tripathi et al. [30] Hygromycin Occupies the ribosome binding site of the elongation factor EF-2 hpt Indica rice MR 219 Agrobacterium-medi- ated transformation Rice callus 20 mg/l Zuraida et al. [36] Hygromycin resistance gene Japonica rice cultivar ‘Nipponbare’ Agrobacterium-medi- ated transformation Rice callus 50 mg/l Toki et al. [34] hptII, Hygromycin resistance gene Indica rice MR219 line 4 and line 9 Agrobacterium- medi- ated transformation Rice callus 45 mg/l Htwe et al. [37] Herbicides l -Phosphatidylserine (PPT) Inhibits glutamine synthetase (GS), a key enzyme in nitrogen assimilation, thus causing ammonia accumulation, glu- tamine depletion and eventually plant death bar or pat, bleomycin acetyl transferase (BAT) Oryza sativa L. (cv Taipei 309) Microprojectile medi- ated transformation Rice embryo tissue 4 mg/l Cao et al. [47] Glyphosate Inhibits Shikimate pathway leading to the accumulation of shikimate thus inhibits the synthesis of aro- matic amino acids and secondary metabolites, causing cell death I.variabilis-EPSPS Japonica rice cultivar Zhonghua11 Agrobacterium-medi- ated transformation Rice callus 200 mg/l Cui et al. [53] Metabolic markers Mannose Plant cells lacking this enzyme cannot survive on synthetic medium containing mannose as a carbon source Pmi gene from E. coli, Phosphomannose isomerase (PMI) Japonica rice var. TP 309 Agrobacterium-medi- ated transformation Rice callus 30 g/l Lucca et al. [56] manA gene from E. coli Japonica, var Ishikari-shiroge Agrobacterium-medi- ated transformation Rice callus 25 g/l He et al. [54] Pmi gene from E. coli Japonica rice var. Zhon- ghua 8 Agrobacterium-medi- ated transformation Rice callus 10 g/l Zai-Song et al. [57]
Molecular Biotechnology 1 3 transformation efficiency by using Agrobacterium medi- ated transformation of Oryza sativa variety, Pusa Basmati-1 (IET-10364) using callus culture and select transformed cal- lus using 50 mg/l kanamycin [30]. Hygromycin, another antibiotic, has been successfully used to select resistant tissue with persistent transgene inheritance in rice [31–34]. The bacterial aph (4′) encoded aminoglycoside phosphotransferase (also known as hygro- mycin phosphotransferase) inactivates hygromycin by phos- phorylating its hydroxyl group [35]. Zuraida et al. employ 10–20 mg/l hygromycin to select indica rice MR 219 [36]. Hygromycin has also been used as a selection marker for rice transformants in other research [34, 37]. Another antibiotic known as Bleomycin has also been employed as SMG in rice studies [38]. In the presence of ­Fe2+ and ­ O2, the interaction of bleomycin with the 4′-car- bon-hydrogen bond of deoxyribose causes single and double strand DNA breaks [39]. Bleomycin resistance genes, blmB or blmA has been identified from Streptomyces verticillus. blmB code for bleomycin acetyl transferase (301 aa) whilst blmA encodes bleomycin binding protein (BLMA,122 aa) respectively. BLMA inactivates bleomycin by binding it non-covalently (both metal-free and metal bound state) with high affinity [40–42]. Whereas, bleomycin acetyl transferase acetylates primary amine group of β-aminoalanine moiety in bleomycin thus rendering it inactive. The metal che- lated complex is prevented from coordinating and reducing molecular oxygen by acetylation. If oxygen free radicals are not suppressed, single-stranded and double-stranded DNA breaks occur, resulting in cell death [19, 43, 44]. Dekeyser et al. has employed 20 mg/l concentration of bleomycin to select rice plants transformants [38]. Herbicides Resistant Genes as SMGs A few herbicide-tolerant genes, such as bar, pat, and aroA, are used as SMGs to select transformed plant tissue. Phos- phinothricin-N-acetyltransferase enzymes encoded by the bar and pat genes of Streptomyces hygroscopicus and Strep- tomyces viridochromogenes acetylate and detoxify phosphi- nothricin. Although, Phosphinothricin-N-acetyltransferase enzymes encoded by these two genes share 85% identity at the amino acid level, they are functionally equivalent [45, 46]. Transgenic lines have already been selected using phos- phinothricin as selection agent. At 4 and 8 mg/l glufosinate ammonium (the ammonium salt of phosphinothricin), Cao et al. observe substantial cell growth retardation in rice sus- pension culture (Oryza sativa L. cv Taipei 309 [47]). Other studies also report the selection of rice transformants using 4 mg/l of glufosinate ammonium [38, 47]. Glyphosate, a herbicide work by competitively binding to 5-enolpyruvyl shikimate 3-phosphate synthase, also called aroA enzyme of shikimate pathway this inhibiting biosyn- thesis of aromatic amino acids. The impaired synthesis of aromatic amino acids and secondary metabolites thus results in plant cell death [48, 49]. In plants, glyphosate resistance has been achieved by introducing genes encoding an EPSPS enzyme with reduced affinity for glyphosate. A modified EPSPS has been employed to achieve enhanced glyphosate resistance in transgenic rice [50, 51]. In lab experiments and field trials, transgenic japonica rice cultivar Xiushui-110 harbouring a novel G6 gene from Pseudomonas putida (encoding EPSPS) tolerates 8 gm/l of glyphosate concen- tration compared to conventional rice sensitive to 1 g/l weed control glyphosate spray dose [52]. Cui et al. develops novel glyphosate tolerant Japonica rice lines by transforming epsps gene from Isoptericola variabilis gene using Agrobacterium mediated transformation method and select resistant calli using 200 mg/l glyphosate [53]. Metabolic Genes as SMGs Many plants, including rice, lacking phosphomannose- isomerase, are unable to grow in the presence of mannose and accumulate it as mannose-6-phosphate in the roots [54]. The accumulation of mannose-6-phosphate inhibits phosphoglucose isomerase and thus glycolysis [55]. Phos- phomannose isomerase enzyme encoded by manA gene of E. coli has been employed in rice crop [54, 56, 57]. manA encoded phosphomannose isomerase catalyses the conver- sion of mannose 6-phosphate to fructose 6-phosphate [25]. He et al. utilise 25 g/l of mannose to select Japonica rice (Oryza sativa L. ssp. Japonica, var. Ishikari-shiroge, I-S) transformants [54]. In another study, Zai-Song employs a combination of 10 g/l of mannose and 5 g/l of glucose for selecting japonica rice variety Zhonghua 8 [57]. Perspectives of Selectable Marker to Marker‑Free Selection in Rice SMG coupled to a transgene gives transformed cells a growth advantage during the transformation process in gen- eral. After achieving successful transformation, these SMGs are no longer required, thus undesirable. Integration of SMG in genetically modified (GM) crops is a fundamental deter- minant of the commercialization of GM plants and their products. It's extremely unlikely that a gene could be hori- zontally transferred from plant products to the gut micro- biota, intestinal cells, the environment, or therapeutically important bacteria. In 2009, China’s Ministry of Agriculture gave biosafety certificates to two transgenic rice cultivars, Huahui No. 1 and Bt Shanyou 63. Both of these cultivars are pest-resistant and carry pest-resistant transgenes. The
Molecular Biotechnology 1 3 hph gene, conferring resistance to hygromycin, has been employed as a selective marker gene in both these cultivars [58]. Furthermore, despite transgenics delivering environ- mental benefits, the study emphasises risk management to restrict gene flow from Bt rice to wild and weedy rice [59]. After long-term intake of GM rice, maize, and potato, an animal study finds little differences in biochemical, haema- tological, and histological assessment when compared to a control group. Because the impacts are non-toxic and fall within the typical variation range, the study concludes that GM foods are nutritionally equivalent to their wild coun- terparts. Wang et al. looked at the effect of the bar gene (an anti-herbicide) in rice transgenics to see if it was safe to eat. Rats fed with transgenic rice for 30 days, show no change in body weight, organs, blood composition, or other pathohistological characteristics, rendering it as safe [60]. Basmati rice is highly prized in India, although it is nutri- tionally deficient. To boost the nutritional value of rice basmati, transgenic studies have been proposed. However, because our country’s rules prohibit the sale of genetically modified foods, a premium crop like Basmati cannot be used in a unique fashion [61]. Herbicide-resistant transgenic rice varieties have been licenced for usage in the United States, although they have yet to be commercialised. India has approved limited field experiments for insect, bacterial blight, fungus, salt, and pest-resistant transgenic rice. To date, severe restrictions and policies control the commer- cialization of transgenic rice and other cereal crops in many nations. The most serious concern about eating GM food is the spread of antibiotic-resistant genes from transgenic plants to bacterial populations, resulting in superbugs. As a result, scientists are concentrating their efforts on reduc- ing genetic load in GM crops and increasing global accept- ance of GM crops by either eradicating SMGs or generating selection marker-free GM crops [62]. SMGs Elimination Strategies Employed in Rice Using Co‑transformation One of the earliest methods for producing transgenics without the selectable marker is the co-transformation. It involves transforming two or more genes (gene of interest and selectable marker gene) simultaneously into the plant genome. Using the same or different Agrobacterium, two T-DNAs can be transformed into a single binary vector or two binary vectors with one gene each [63–65]. Transfor- mation using Agrobacterium and particle bombardment are equally efficient to achieve desired results. However, particle bombardment leads to the co-integration of T-DNAs at the same genomic locus resulting in the linkage between gene of interest and marker gene [66]. It requires multiple genera- tions to remove the SMG, as it gets often linked with genes. Further, this method is ineffective for transgenic trees with longer generation time period and sterile plants. Parkhi et al. utilizes two binary plasmids to successfully generate marker- free carotenoids-rich transgenic rice (containing psy and crtI gene encoding phytoene synthase and phytoene desaturase respectively). The study reports 53.9% co-transformation efficiency in transgenic rice employing hph and nptII as selectable marker and gus as scorable marker [67]. Sripriya et al., develops marker-free sheath blight resistant rice plant carrying chitinase (chi11) gene using same method. To pro- duce marker-free rice plants, an Agrobacterium strain with a binary vector carrying the gene of interest (chi11) cloned under the maize ubiquitin promoter and a co-integration vec- tor with the marker gene (hph) is co-transformed. With a co-transformation efficiency of 20%, segregation of the gene of interest and the marker gene is achieved in ­ T1 generation [68]. Ramana Rao et al. employ modified co-transformation method to generate marker-free transgenic sheath blight resistant rice cultivar by introducing two genes, chi11 and ap24. chi11 encodes endochitinase whereas ap24 encodes osmotin exhibiting antifungal activity against Phytoph- thora infestans. ­T1 generation reveals effective separation of SMG and gene of interest to different genetic loci with a co-transformation efficiency of 67% [69]. The approach has also been employed to generate marker-free insect resistant transgenic Indica rice using hpt as SMG [70]. Using Site‑Specific Recombination Site-specific recombination is the exchange of genetic mate- rial between pairs of short, defined sequences at certain sites [71]. In plants, numerous types of site-specific recombina- tion systems have been employed to achieve marker-free transgenics. These include CRE-loxP system from P1 bac- teriophage, R/RS system from Zygosaccharomyces rouxii [72–74] (Fig.1) and FLP/FRT from yeast, Saccharomyces cerevisiae [75]. CRE-loxP system includes a CRE recom- binase, and loxP recognition site. Two 13-bp loxP palindro- mic sequences flanking a 7–12 bp core sequence are recog- nised by CRE recombinase [76]. Site-specific recombinase enzyme cleaves DNA at borders between recombinase bind- ing elements and core sequence. CRE recombinase enzyme recognise loxP recognition sites (flanking the selectable marker gene) and execute DNA excision and recombina- tion. CRE-loxP system has been used to develop marker-free transgenic tobacco and rice by removing hpt gene [77, 78]. Sreekala et al. employ a chemically regulated CRE-loxP- mediated site-specific recombination technique to create marker-free rice transgenic plants in a single transforma- tion. Out of 86 separate transgenic lines, 10 plants in the ­ T0 generation and 17 in T1 generation successfully segregate as
Molecular Biotechnology 1 3 marker-free plants [78]. Bai et al. generate marker-free trans- genic rice by employing CRE-loxP system (with the cre gene expressed under the Osmads45 promoter) and excising nptII gene flanked by lox recombination sites at ­ T1 stage with 37.5% auto excision efficiency [79]. Sengupta et al. generate marker-free transgenic rice resistant to sap sucking plan- thoppers using CRE-loxP recombination technology. The hpt marker gene cassette is cloned between the loxP sites in vector harbouring Allium sativum leaf agglutinin encoding gene. cre gene is cloned in a separate vector. Reciprocal crosses between single-copy ­ T0 plants harbouring cre-bar T-DNA and single-copy ­ T0 plants harbouring Allium sati- vum leaf agglutinin -lox-hpt-lox T-DNA results in marker- free ­T1 hybrids. The homozygous rice lines get subsequently established in ­ T3 generation [80]. cre gene expressed under the soybean heat-shock pro- moter in Nipponbare rice has been marked as an effective strategy for conditional removal of marker gene in seed- ling upon heat treatment [81]. Radhakrishnan and Srivas- tava establishes FLP/FRT recombination system for rice by deleting hpt gene from transformed plants [82]. FLP/ FRT recombination system has also been used to remove nptII from GM rice [83]. Woo et al. develop marker-free transgenic rice using an oxidative stress inducible FLP/FRT based recombination system employing ­ H2O2. The system involves a binary vector carrying codon optimized mFLP (modified S. cerevisiae FLP with GC content) and hpt (as SMG) between two FRT sites. The oxidative stress inducible peroxidase promoter from sweet potato is used to express both SMG and mFLP genes. Increased level of tocopherol (due to overexpression of NtTc, Nicotiana tabacum cultivar Xanthi tocopherol cyclase) assist in selection of transgen- ics callus followed by segregation of marker gene by auto excision. Compared to other methods, this technique elimi- nates the need of additional chemical treatment or crossing with recombinases to remove marker gene from transformed rice plants [84]. Nandy and Srivastava develop marker-free transgenic rice in ­ T0 generation by site-specific integra- tion using FLP/FRT and CRE-loxP for marker elimination. Marker-free site-specific integration (MF-SSI) is observed in the first generation transgenic rice plant. CRE-loxP medi- ated method to generate marker-free crop is most efficient and stable method to transmit MF-SSI locus to next genera- tion thereby producing marker-free first generation progeny [85]. In another study thermostable FLP recombinase have shown 100% excision efficiency in transgenic rice com- pared to wild-type FLP recombinase (FLPwt), implying it a significant step towards FLP-FRT based biotechnology in plants [86]. Using Transposable Elements Transposable elements are 100–1000 bases long sequences involved in DNA repositioning in a genome. Barbara McClintock identifies the first transposons, Ac/Ds family in maize. Ac stands for activator element encoding transposase and Ds stands for dissociation element. Transposase helps to mobilize marker gene cloned between the inverted repeats of Ds [87]. After the expression of the transposase, transpos- able elements can be excised and re-insert in the genome. The marker and gene of interest can be separated by plac- ing the marker gene or gene of interest within the jumping sequence. Because the marker gene is genetically unlinked from the gene of interest, marker-free plants can be selected after segregation. Due to the spectrum of positional effects caused by re-insertion, a single transpositionally active Fig. 1  SMG removal by site-specific recombination system. LB left border, T terminator, S selectable marker gene, P promoter, G gene of inter- est, iP inducible promoter, R recombinase gene, RB right border [98]
Molecular Biotechnology 1 3 transformant line can demonstrate substantial qualitative and quantitative diversity in gene expression levels [88]. However, reinsertion phenomenon can lower the impact of marker gene removal and may enhance genome instability in transgenics due to deletions, inverted duplications, inver- sions, and translocations [89]. Cotsaftis et al. successfully develop marker-free trans- genic Bt rice using transposable element-based approach. This rice transformation study involves T-DNA carrying cry1B gene under maize ubiquitin promoter flanked by inverted terminal repeats of Ds cloned in the 5′untranslated sequence of green fluorescent protein gene, an Ac trans- posase and hph gene expressed under CaMV35S promoter. The successful production of T-DNA free lines as a result of Ds-cry1B relocation in the rice genome suggests this as potent strategy for generating T-DNA free transgenic plants [88]. Gao et al. also generate marker-free Bt-δ endotoxin transgenic rice employing maize Ds element. The green fluorescent protein gene acts as counter marker. Marker-free progeny are identified in ­ T1 generation following unlinked germinal transposition in 26.1% of primary transformants. However, the method is time consuming, labour intensive, as it requires crossing of transgenic plants and the selec- tion of the marker-free progeny. This further depends on the recovery rate of unlinked germinal transposition. Further, probability of recombination can be enhanced by increasing the population size of ­ T1 generation [90]. CRISPR/Cas9 Gene editing or manipulation has become much easier with discovery of CRISPR/Cas9 system. The RNA based sys- tem utilizes clustered regularly interspaced short palindro- mic repeats (CRISPR) and Cas9 nuclease for editing tar- geted gene. A simple guide RNA complementary to target sequence scans the target DNA for a protospacer adjacent motif [91]. For optimized gene expression, codon optimized Cas9 is available for rice. CRISPR/Cas9 system has already been employed for trait modification in O. sativa. Recently, Srivastava et al. employs bi-allelic CRISPR based meth- odology for excising gus (β-glucuronidase) gene from rice genomes with precise cut and ligation of two blunt ends of the gene. This method detected no mutation at or around the excision site, a highly required goal of marker-removal technologies for precise and unaltered excision of and around target DNA [92]. Molecular analysis further reveals significant excision frequency of this system amongst plant lines compared to callus lines. Lu et al. propose the applica- tion of CRISPR/Cas9 technology to promoter editing [93]. Wu et al. develop ­ H2O2 and 3,3-Diaminobenzidine based high-throughput visual detection method to verify CRISPR/ Cas9 edited transgene-free rice plants. This study employ polymerase chain reaction and 3,3-Diaminobenzidine stain- ing to detect the difference in ­ H2O2 levels in transgenic rice plant containing hpt gene from non-transgenic [94]. Toda et al. attempt direct delivery of Cas9-gRNAs ribonucleopro- teins (targeting DsRed2) into rice zygotes in transgenic rice plants expressing DsRed2. DsRed2 expression is reduced in zygotes and/or subsequent embryos when sgRNA in ribo- nucleoproteins targets the DsRed2 sequence. As the method uses plant zygotes it does not require selectable marker gene to edit plant genome. Zygotes with decreased DsRed2 signal regenerate in to mature plant in the absence of any selection agents, with 14–64% showing targeted mutations. This study highlights the enormous potential of this method in other important crop too such as maize and wheat [95]. Dong et al. develop marker-free transgenic carotenoid rich rice using CRISPR/Cas9. The 5.2 kb marker-free carotenoid cas- sette is inserted at genomic safe harbour regions in the plant genome. Transformed dehusked seeds develop golden colour due to the presence of carotenoids in endosperm compared to wild type seeds [96]. Li et al. develop blight resistant transgenic free rice. In this study, partial sequence of Xa13 promoter was edited by CRISPR/Cas9. This does not alter the gene expression and function but alter its ability to get induced by pathogen. This editing improves the rice abil- ity to resist disease without affecting its fertility. PCR was used to identify the mutation site induced by double target sgRNA [97]. Conclusion Rice, after wheat, is the world’s second-largest source of human food energy. Innovative crop improvement technolo- gies are need of the hour to meet the growing food demand of the world’s population. This research focuses on advance- ments in rice transgenics for enhanced traits, with a focus on selection approaches that range from the use of existing SMGs to selectable marker-free progenies. Popular first gen- eration SMGs including hph, hpt, and nptII are regarded as threat to humanity if not removed before reaching the con- sumer. Second-generation selectable markers like as manA and xylA have not yet been discovered as possibly detrimen- tal to the environment, but they are still unappealing to con- sumers. As a result, transgene-free plants are the most attrac- tive and commercially acceptable. Since the first transgenic crop was released in 1996, there has been an 87-fold increase in the number of GM crops adopted around the world. Sev- eral ways in combination with the CRISPR/Cas9 editing tool, have been demonstrated as beneficial in rice genome editing. This have been achieved either by removing SMGs or by genome editing without the need of transgene integra- tion. However, no such GM crop has yet been sold, leaving plenty of room for experimentation. Similar rice engineering
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Selectable Markers to Marker‑Free Selection in Rice

  • 1. Vol.:(0123456789) 1 3 Molecular Biotechnology https://doi.org/10.1007/s12033-022-00460-w REVIEW Selectable Markers to Marker‑Free Selection in Rice Aditi Sharma1 · Ayush Chouhan1 · Tarun Bhatt1 · Anupreet Kaur1 · Anu Priya Minhas1 Received: 21 August 2021 / Accepted: 3 February 2022 © The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2022 Abstract Inadequate rice production worldwide is largely attributed to abiotic and biotic stresses, along with high sensitivity of cultivable plant germplasm. In the field of cereal biotechnology, rice engineering plays an important role in achieving tol- erance to such stresses. Plant transformation and selection play crucial role in rice engineering. This review summarized the antibiotic, herbicide and metabolic selection marker genes (SMG) employed in diverse rice engineering studies. These SMGs are no longer required after the transformation has been achieved, hence undesirable at the commercial level. This study also included several strategies employed in rice engineering to eliminate such foreign DNA elements. These include co-transformation, site-specific recombination, transposon and CRISPR base approaches. CRISPR/Cas9 being simple and efficient, is considered a crucial step toward clean gene technology. Further ease and applicability of CRISPR/Cas9 in the embryos directly can help us to modify target genes with efficient marker-free selection in minimum time. Overall, this review summarizes and analyse the recent advances that have enormous potential in rice improvement. Keywords Rice · Selection marker gene · Marker-free selection · CRISPR · Transformation · Engineering Abbreviations SMG Selectable marker gene nptII Neomycin phosphotransferase II encoding gene EPSPS 5-Enolpyruvyl shikimate 3-phosphate synthase hpt Hygromycin phosphotransferase encoding gene GM Genetically modified hph Hygromycin B phosphotransferase encoding gene T-DNA Transfer DNA FLP/FRT Flippase/FLP recombinase recognition target CRE/loxP Cre recombinase/locus of x-over, P1 CRISPR Clustered regularly interspaced short palin- dromic repeats Cas CRISPR associated protein Introduction Rice (Oryza sativa L.) is the second most cultivated cereal crop worldwide [1]. Interestingly, every second person on this planet relies on rice as the staple food. Asia alone holds the major share (above 90%) of world’s rice production and consumption. There are 24 species in the genus Oryza, only two are domesticated. Oryza sativa is an Asian rice, widely cultivated in southern and eastern Asia. The African rice, Oryza glaberrima, is native to and confined mostly to western Africa. O. sativa, O. glaberrima and their wild progenitors are diploids (2n=24) with AA genomes [2, 3]. In the past, O. sativa has been divided into two subspecies, Japonica and Indica, but recent genetic studies suggest five distinct groups of rice [4]. From 1996 to 2011, rice produc- tion rose by 180% from 257 million tons to 718 million tons owing to the green revolution. However, population explo- sion in low-income countries with 800 million people has Aditi Sharma and Ayush Chouhan have contributed equally. * Anu Priya Minhas annuminhas@gmail.com Aditi Sharma aditisharma102@gmail.com Ayush Chouhan ayushc125@gmail.com Tarun Bhatt tarunbhatt0300@gmail.com Anupreet Kaur anupreetz@yahoo.co.in 1 Department of Biotechnology, University Institute of Engineering and Technology, Sector‑25, Panjab University, Chandigarh 160014, India
  • 2. Molecular Biotechnology 1 3 paralyzed us to get sufficient food for everyone. The ultimate vision for 2050 is to produce staple foods on the same piece of arable land for this increased population [5, 6]. Need of Genetic Engineering in Rice Crop In rice crop, many promising varieties have the potential to produce 10 ton per hectare yield under controlled environ- ment. However, local farmers under field conditions end up harvesting half of the actual yield reasoned to various envi- ronmental stresses [7]. Drought, salinity, submergence, cold, high temperature, and metal toxicity are examples of biotic (insect and pest) and abiotic (drought, salinity, submergence, cold, high temperature, and metal toxicity) environmental stresses. Drought stress is one of the abiotic stresses that poses a severe threat to world rice production when water is scarce. Drought stress alone has been estimated to reduce worldwide rice production by 18 million tons annually. It affects plant growth by reducing cell growth, cell elonga- tion, and cell expansion. As a result, reactive oxygen species build up, damaging the plant's antioxidant system. By regu- lating stress-induced gene and protein functions, drought stress modifies morphological, biochemical, physiological, and molecular responses. Salt concentrations in the soil is another crucial factor affecting rice plant development at all stages by causing ionic or osmotic stress [8–10]. Excess of sodium ions compete with useful ions and upset the ionic balance. Osmotic stress can influence the plant’s ability to absorb water from the soil. Reduction in cellular water potential due to the increased solute concentration affects stomatal conductance, transpiration, gaseous exchange and rate of carbon assimilation [11, 12]. Complete submer- gence also affects many upland rice cultivars critically. A moderate waterlogged environment can extend leaves and stems, resulting in excessive energy use and death of cells [13, 14]. Low temperatures also affect rice germination, seedling growth, leaf curving, shoot length, flowering and tillering [15, 16]. Further, due to the limited variety of cul- tivable rice cultivars and the labour intensive agriculture techniques, rice cultivation is seasonal. In order to develop varieties resistant to these stresses, it is essential to under- stand how plants respond to these stresses. Biggest challenge in plant biotechnology is to develop stress tolerant plants employing genetically engineered technology. Traditional crop improvement practices such as plant breeding have their own limits. At present, genetic engineering is utilized to increase the genetic pool of crop species. As a result, genes from a variety of organisms can be introduced with new or enhanced functions when exposed to certain biotic and abi- otic stresses. Being quick, accurate, and stable, genetic engi- neering is speculated as superior alternative method. How- ever, exact selection of true transgenics with high precision depends on rigorous selection procedures. Through the transfer of desirable genes, this technology allows access to an unlimited gene pool. The transformation method quiet efficient to produce useful plants with special phenotypes within a short period of time, rectify faults, and improve physiological and agronomical traits [5]. DNA introduction and selection of true transformed plant cells/tissue constitute two most demanding steps of rice transformation [17]. Rice plant transformation involves introducing foreign DNA seg- ments with a selectable marker gene (SMG) into either plant nuclear DNA or chloroplast genome [18]. Proteins encoded by SMGs confer competitive advantage on only transformed cells, making them more likely to be selected in presence of a selection agent over untransformed cells. SMGs Antibiotic/herbicide resistance genes are initially employed for successfully selecting transgenic model plants resistant to various biotic and abiotic traits. Later, the same technology has been extended to crops including rice and other cere- als successfully [19, 20] (Table1). Transformed cells show resistance to antibiotics when antibiotic resistance genes from bacterial origin are expressed under plant-specific pro- moters. Glycopeptides and aminoglycosides constitute major classes of antibiotics employed in plant research. Hygromy- cin, kanamycin, neomycin, gentamicin, geneticin (G418) and paromomycin are of aminoglycoside types whereas bleomy- cin belongs to glycopeptide family [21]. Antibiotics in the aminoglycoside family work as protein inhibitors, halting cell growth [22]. Kanamycin, gentamicin, geneticin and neomycin block protein synthesis by binding 30S subunit in ribosome whilst hygromycin occupies the ribosome binding site in the elongation factor EF-2 [23]. Plants’ sensitivity, on the other hand, varies greatly between species, genotypes, and tissues [24–27]. Antibiotic Resistant Genes as SMGs Neomycin phosphotransferase II (aphA2), expressed by EPSPS gene, from Escherichia coli transfer phosphate molecule to their respective hydroxyl groups and confers resistance to kanamycin and G418. The phosphate molecule hinders with the interactions and binding of antibiotics to ribosomes. Same has also been employed as a selection marker in rice as well. Kanamycin (150 mg/l) along with cefotaxime has been used for selecting Indica rice (Oryza sativa L.) cv. RD6 transformants [28]. Chan et al. report the transformation of Indica rice cv. Taichung Native 1 using nptII gene and select the transformed rice using 20 mg/l of kanamycin [29]. Tripathi et al. attempt to increase
  • 3. Molecular Biotechnology 1 3 Table 1  List of rice transformation studies indicating their corresponding SMG, selection agents, mode of action, transformation method, plant part used and MIC of the selection agent Selection agent Mode of action Resistance gene and encoded enzyme Rice used in the study Transformation tech- nique used Plant part used MIC value of selection agent References Aminoglycosides Kanamycin, Geneticin (G418) Binds to the 30S riboso- mal subunit nptII, Neomycin phosphotransferase II enzyme (NPT) Oryza sativa L Electroporation Rice protoplast 200 mg/l Dekeyser et al. [38] (Oryza sativa L.) cv. RD6 Agrobacterium-medi- ated transformation Rice callus 150 mg/l Thodsaporn et al. [28] Indica rice cv. Taichung native 1 Agrobacterium-medi- ated transformation Rice callus 20 mg/l Chan et al. [29] Pusa Basmati-1 (IET- 10364) Agrobacterium-medi- ated transformation Rice callus 50 mg/l Tripathi et al. [30] Hygromycin Occupies the ribosome binding site of the elongation factor EF-2 hpt Indica rice MR 219 Agrobacterium-medi- ated transformation Rice callus 20 mg/l Zuraida et al. [36] Hygromycin resistance gene Japonica rice cultivar ‘Nipponbare’ Agrobacterium-medi- ated transformation Rice callus 50 mg/l Toki et al. [34] hptII, Hygromycin resistance gene Indica rice MR219 line 4 and line 9 Agrobacterium- medi- ated transformation Rice callus 45 mg/l Htwe et al. [37] Herbicides l -Phosphatidylserine (PPT) Inhibits glutamine synthetase (GS), a key enzyme in nitrogen assimilation, thus causing ammonia accumulation, glu- tamine depletion and eventually plant death bar or pat, bleomycin acetyl transferase (BAT) Oryza sativa L. (cv Taipei 309) Microprojectile medi- ated transformation Rice embryo tissue 4 mg/l Cao et al. [47] Glyphosate Inhibits Shikimate pathway leading to the accumulation of shikimate thus inhibits the synthesis of aro- matic amino acids and secondary metabolites, causing cell death I.variabilis-EPSPS Japonica rice cultivar Zhonghua11 Agrobacterium-medi- ated transformation Rice callus 200 mg/l Cui et al. [53] Metabolic markers Mannose Plant cells lacking this enzyme cannot survive on synthetic medium containing mannose as a carbon source Pmi gene from E. coli, Phosphomannose isomerase (PMI) Japonica rice var. TP 309 Agrobacterium-medi- ated transformation Rice callus 30 g/l Lucca et al. [56] manA gene from E. coli Japonica, var Ishikari-shiroge Agrobacterium-medi- ated transformation Rice callus 25 g/l He et al. [54] Pmi gene from E. coli Japonica rice var. Zhon- ghua 8 Agrobacterium-medi- ated transformation Rice callus 10 g/l Zai-Song et al. [57]
  • 4. Molecular Biotechnology 1 3 transformation efficiency by using Agrobacterium medi- ated transformation of Oryza sativa variety, Pusa Basmati-1 (IET-10364) using callus culture and select transformed cal- lus using 50 mg/l kanamycin [30]. Hygromycin, another antibiotic, has been successfully used to select resistant tissue with persistent transgene inheritance in rice [31–34]. The bacterial aph (4′) encoded aminoglycoside phosphotransferase (also known as hygro- mycin phosphotransferase) inactivates hygromycin by phos- phorylating its hydroxyl group [35]. Zuraida et al. employ 10–20 mg/l hygromycin to select indica rice MR 219 [36]. Hygromycin has also been used as a selection marker for rice transformants in other research [34, 37]. Another antibiotic known as Bleomycin has also been employed as SMG in rice studies [38]. In the presence of ­Fe2+ and ­ O2, the interaction of bleomycin with the 4′-car- bon-hydrogen bond of deoxyribose causes single and double strand DNA breaks [39]. Bleomycin resistance genes, blmB or blmA has been identified from Streptomyces verticillus. blmB code for bleomycin acetyl transferase (301 aa) whilst blmA encodes bleomycin binding protein (BLMA,122 aa) respectively. BLMA inactivates bleomycin by binding it non-covalently (both metal-free and metal bound state) with high affinity [40–42]. Whereas, bleomycin acetyl transferase acetylates primary amine group of β-aminoalanine moiety in bleomycin thus rendering it inactive. The metal che- lated complex is prevented from coordinating and reducing molecular oxygen by acetylation. If oxygen free radicals are not suppressed, single-stranded and double-stranded DNA breaks occur, resulting in cell death [19, 43, 44]. Dekeyser et al. has employed 20 mg/l concentration of bleomycin to select rice plants transformants [38]. Herbicides Resistant Genes as SMGs A few herbicide-tolerant genes, such as bar, pat, and aroA, are used as SMGs to select transformed plant tissue. Phos- phinothricin-N-acetyltransferase enzymes encoded by the bar and pat genes of Streptomyces hygroscopicus and Strep- tomyces viridochromogenes acetylate and detoxify phosphi- nothricin. Although, Phosphinothricin-N-acetyltransferase enzymes encoded by these two genes share 85% identity at the amino acid level, they are functionally equivalent [45, 46]. Transgenic lines have already been selected using phos- phinothricin as selection agent. At 4 and 8 mg/l glufosinate ammonium (the ammonium salt of phosphinothricin), Cao et al. observe substantial cell growth retardation in rice sus- pension culture (Oryza sativa L. cv Taipei 309 [47]). Other studies also report the selection of rice transformants using 4 mg/l of glufosinate ammonium [38, 47]. Glyphosate, a herbicide work by competitively binding to 5-enolpyruvyl shikimate 3-phosphate synthase, also called aroA enzyme of shikimate pathway this inhibiting biosyn- thesis of aromatic amino acids. The impaired synthesis of aromatic amino acids and secondary metabolites thus results in plant cell death [48, 49]. In plants, glyphosate resistance has been achieved by introducing genes encoding an EPSPS enzyme with reduced affinity for glyphosate. A modified EPSPS has been employed to achieve enhanced glyphosate resistance in transgenic rice [50, 51]. In lab experiments and field trials, transgenic japonica rice cultivar Xiushui-110 harbouring a novel G6 gene from Pseudomonas putida (encoding EPSPS) tolerates 8 gm/l of glyphosate concen- tration compared to conventional rice sensitive to 1 g/l weed control glyphosate spray dose [52]. Cui et al. develops novel glyphosate tolerant Japonica rice lines by transforming epsps gene from Isoptericola variabilis gene using Agrobacterium mediated transformation method and select resistant calli using 200 mg/l glyphosate [53]. Metabolic Genes as SMGs Many plants, including rice, lacking phosphomannose- isomerase, are unable to grow in the presence of mannose and accumulate it as mannose-6-phosphate in the roots [54]. The accumulation of mannose-6-phosphate inhibits phosphoglucose isomerase and thus glycolysis [55]. Phos- phomannose isomerase enzyme encoded by manA gene of E. coli has been employed in rice crop [54, 56, 57]. manA encoded phosphomannose isomerase catalyses the conver- sion of mannose 6-phosphate to fructose 6-phosphate [25]. He et al. utilise 25 g/l of mannose to select Japonica rice (Oryza sativa L. ssp. Japonica, var. Ishikari-shiroge, I-S) transformants [54]. In another study, Zai-Song employs a combination of 10 g/l of mannose and 5 g/l of glucose for selecting japonica rice variety Zhonghua 8 [57]. Perspectives of Selectable Marker to Marker‑Free Selection in Rice SMG coupled to a transgene gives transformed cells a growth advantage during the transformation process in gen- eral. After achieving successful transformation, these SMGs are no longer required, thus undesirable. Integration of SMG in genetically modified (GM) crops is a fundamental deter- minant of the commercialization of GM plants and their products. It's extremely unlikely that a gene could be hori- zontally transferred from plant products to the gut micro- biota, intestinal cells, the environment, or therapeutically important bacteria. In 2009, China’s Ministry of Agriculture gave biosafety certificates to two transgenic rice cultivars, Huahui No. 1 and Bt Shanyou 63. Both of these cultivars are pest-resistant and carry pest-resistant transgenes. The
  • 5. Molecular Biotechnology 1 3 hph gene, conferring resistance to hygromycin, has been employed as a selective marker gene in both these cultivars [58]. Furthermore, despite transgenics delivering environ- mental benefits, the study emphasises risk management to restrict gene flow from Bt rice to wild and weedy rice [59]. After long-term intake of GM rice, maize, and potato, an animal study finds little differences in biochemical, haema- tological, and histological assessment when compared to a control group. Because the impacts are non-toxic and fall within the typical variation range, the study concludes that GM foods are nutritionally equivalent to their wild coun- terparts. Wang et al. looked at the effect of the bar gene (an anti-herbicide) in rice transgenics to see if it was safe to eat. Rats fed with transgenic rice for 30 days, show no change in body weight, organs, blood composition, or other pathohistological characteristics, rendering it as safe [60]. Basmati rice is highly prized in India, although it is nutri- tionally deficient. To boost the nutritional value of rice basmati, transgenic studies have been proposed. However, because our country’s rules prohibit the sale of genetically modified foods, a premium crop like Basmati cannot be used in a unique fashion [61]. Herbicide-resistant transgenic rice varieties have been licenced for usage in the United States, although they have yet to be commercialised. India has approved limited field experiments for insect, bacterial blight, fungus, salt, and pest-resistant transgenic rice. To date, severe restrictions and policies control the commer- cialization of transgenic rice and other cereal crops in many nations. The most serious concern about eating GM food is the spread of antibiotic-resistant genes from transgenic plants to bacterial populations, resulting in superbugs. As a result, scientists are concentrating their efforts on reduc- ing genetic load in GM crops and increasing global accept- ance of GM crops by either eradicating SMGs or generating selection marker-free GM crops [62]. SMGs Elimination Strategies Employed in Rice Using Co‑transformation One of the earliest methods for producing transgenics without the selectable marker is the co-transformation. It involves transforming two or more genes (gene of interest and selectable marker gene) simultaneously into the plant genome. Using the same or different Agrobacterium, two T-DNAs can be transformed into a single binary vector or two binary vectors with one gene each [63–65]. Transfor- mation using Agrobacterium and particle bombardment are equally efficient to achieve desired results. However, particle bombardment leads to the co-integration of T-DNAs at the same genomic locus resulting in the linkage between gene of interest and marker gene [66]. It requires multiple genera- tions to remove the SMG, as it gets often linked with genes. Further, this method is ineffective for transgenic trees with longer generation time period and sterile plants. Parkhi et al. utilizes two binary plasmids to successfully generate marker- free carotenoids-rich transgenic rice (containing psy and crtI gene encoding phytoene synthase and phytoene desaturase respectively). The study reports 53.9% co-transformation efficiency in transgenic rice employing hph and nptII as selectable marker and gus as scorable marker [67]. Sripriya et al., develops marker-free sheath blight resistant rice plant carrying chitinase (chi11) gene using same method. To pro- duce marker-free rice plants, an Agrobacterium strain with a binary vector carrying the gene of interest (chi11) cloned under the maize ubiquitin promoter and a co-integration vec- tor with the marker gene (hph) is co-transformed. With a co-transformation efficiency of 20%, segregation of the gene of interest and the marker gene is achieved in ­ T1 generation [68]. Ramana Rao et al. employ modified co-transformation method to generate marker-free transgenic sheath blight resistant rice cultivar by introducing two genes, chi11 and ap24. chi11 encodes endochitinase whereas ap24 encodes osmotin exhibiting antifungal activity against Phytoph- thora infestans. ­T1 generation reveals effective separation of SMG and gene of interest to different genetic loci with a co-transformation efficiency of 67% [69]. The approach has also been employed to generate marker-free insect resistant transgenic Indica rice using hpt as SMG [70]. Using Site‑Specific Recombination Site-specific recombination is the exchange of genetic mate- rial between pairs of short, defined sequences at certain sites [71]. In plants, numerous types of site-specific recombina- tion systems have been employed to achieve marker-free transgenics. These include CRE-loxP system from P1 bac- teriophage, R/RS system from Zygosaccharomyces rouxii [72–74] (Fig.1) and FLP/FRT from yeast, Saccharomyces cerevisiae [75]. CRE-loxP system includes a CRE recom- binase, and loxP recognition site. Two 13-bp loxP palindro- mic sequences flanking a 7–12 bp core sequence are recog- nised by CRE recombinase [76]. Site-specific recombinase enzyme cleaves DNA at borders between recombinase bind- ing elements and core sequence. CRE recombinase enzyme recognise loxP recognition sites (flanking the selectable marker gene) and execute DNA excision and recombina- tion. CRE-loxP system has been used to develop marker-free transgenic tobacco and rice by removing hpt gene [77, 78]. Sreekala et al. employ a chemically regulated CRE-loxP- mediated site-specific recombination technique to create marker-free rice transgenic plants in a single transforma- tion. Out of 86 separate transgenic lines, 10 plants in the ­ T0 generation and 17 in T1 generation successfully segregate as
  • 6. Molecular Biotechnology 1 3 marker-free plants [78]. Bai et al. generate marker-free trans- genic rice by employing CRE-loxP system (with the cre gene expressed under the Osmads45 promoter) and excising nptII gene flanked by lox recombination sites at ­ T1 stage with 37.5% auto excision efficiency [79]. Sengupta et al. generate marker-free transgenic rice resistant to sap sucking plan- thoppers using CRE-loxP recombination technology. The hpt marker gene cassette is cloned between the loxP sites in vector harbouring Allium sativum leaf agglutinin encoding gene. cre gene is cloned in a separate vector. Reciprocal crosses between single-copy ­ T0 plants harbouring cre-bar T-DNA and single-copy ­ T0 plants harbouring Allium sati- vum leaf agglutinin -lox-hpt-lox T-DNA results in marker- free ­T1 hybrids. The homozygous rice lines get subsequently established in ­ T3 generation [80]. cre gene expressed under the soybean heat-shock pro- moter in Nipponbare rice has been marked as an effective strategy for conditional removal of marker gene in seed- ling upon heat treatment [81]. Radhakrishnan and Srivas- tava establishes FLP/FRT recombination system for rice by deleting hpt gene from transformed plants [82]. FLP/ FRT recombination system has also been used to remove nptII from GM rice [83]. Woo et al. develop marker-free transgenic rice using an oxidative stress inducible FLP/FRT based recombination system employing ­ H2O2. The system involves a binary vector carrying codon optimized mFLP (modified S. cerevisiae FLP with GC content) and hpt (as SMG) between two FRT sites. The oxidative stress inducible peroxidase promoter from sweet potato is used to express both SMG and mFLP genes. Increased level of tocopherol (due to overexpression of NtTc, Nicotiana tabacum cultivar Xanthi tocopherol cyclase) assist in selection of transgen- ics callus followed by segregation of marker gene by auto excision. Compared to other methods, this technique elimi- nates the need of additional chemical treatment or crossing with recombinases to remove marker gene from transformed rice plants [84]. Nandy and Srivastava develop marker-free transgenic rice in ­ T0 generation by site-specific integra- tion using FLP/FRT and CRE-loxP for marker elimination. Marker-free site-specific integration (MF-SSI) is observed in the first generation transgenic rice plant. CRE-loxP medi- ated method to generate marker-free crop is most efficient and stable method to transmit MF-SSI locus to next genera- tion thereby producing marker-free first generation progeny [85]. In another study thermostable FLP recombinase have shown 100% excision efficiency in transgenic rice com- pared to wild-type FLP recombinase (FLPwt), implying it a significant step towards FLP-FRT based biotechnology in plants [86]. Using Transposable Elements Transposable elements are 100–1000 bases long sequences involved in DNA repositioning in a genome. Barbara McClintock identifies the first transposons, Ac/Ds family in maize. Ac stands for activator element encoding transposase and Ds stands for dissociation element. Transposase helps to mobilize marker gene cloned between the inverted repeats of Ds [87]. After the expression of the transposase, transpos- able elements can be excised and re-insert in the genome. The marker and gene of interest can be separated by plac- ing the marker gene or gene of interest within the jumping sequence. Because the marker gene is genetically unlinked from the gene of interest, marker-free plants can be selected after segregation. Due to the spectrum of positional effects caused by re-insertion, a single transpositionally active Fig. 1  SMG removal by site-specific recombination system. LB left border, T terminator, S selectable marker gene, P promoter, G gene of inter- est, iP inducible promoter, R recombinase gene, RB right border [98]
  • 7. Molecular Biotechnology 1 3 transformant line can demonstrate substantial qualitative and quantitative diversity in gene expression levels [88]. However, reinsertion phenomenon can lower the impact of marker gene removal and may enhance genome instability in transgenics due to deletions, inverted duplications, inver- sions, and translocations [89]. Cotsaftis et al. successfully develop marker-free trans- genic Bt rice using transposable element-based approach. This rice transformation study involves T-DNA carrying cry1B gene under maize ubiquitin promoter flanked by inverted terminal repeats of Ds cloned in the 5′untranslated sequence of green fluorescent protein gene, an Ac trans- posase and hph gene expressed under CaMV35S promoter. The successful production of T-DNA free lines as a result of Ds-cry1B relocation in the rice genome suggests this as potent strategy for generating T-DNA free transgenic plants [88]. Gao et al. also generate marker-free Bt-δ endotoxin transgenic rice employing maize Ds element. The green fluorescent protein gene acts as counter marker. Marker-free progeny are identified in ­ T1 generation following unlinked germinal transposition in 26.1% of primary transformants. However, the method is time consuming, labour intensive, as it requires crossing of transgenic plants and the selec- tion of the marker-free progeny. This further depends on the recovery rate of unlinked germinal transposition. Further, probability of recombination can be enhanced by increasing the population size of ­ T1 generation [90]. CRISPR/Cas9 Gene editing or manipulation has become much easier with discovery of CRISPR/Cas9 system. The RNA based sys- tem utilizes clustered regularly interspaced short palindro- mic repeats (CRISPR) and Cas9 nuclease for editing tar- geted gene. A simple guide RNA complementary to target sequence scans the target DNA for a protospacer adjacent motif [91]. For optimized gene expression, codon optimized Cas9 is available for rice. CRISPR/Cas9 system has already been employed for trait modification in O. sativa. Recently, Srivastava et al. employs bi-allelic CRISPR based meth- odology for excising gus (β-glucuronidase) gene from rice genomes with precise cut and ligation of two blunt ends of the gene. This method detected no mutation at or around the excision site, a highly required goal of marker-removal technologies for precise and unaltered excision of and around target DNA [92]. Molecular analysis further reveals significant excision frequency of this system amongst plant lines compared to callus lines. Lu et al. propose the applica- tion of CRISPR/Cas9 technology to promoter editing [93]. Wu et al. develop ­ H2O2 and 3,3-Diaminobenzidine based high-throughput visual detection method to verify CRISPR/ Cas9 edited transgene-free rice plants. This study employ polymerase chain reaction and 3,3-Diaminobenzidine stain- ing to detect the difference in ­ H2O2 levels in transgenic rice plant containing hpt gene from non-transgenic [94]. Toda et al. attempt direct delivery of Cas9-gRNAs ribonucleopro- teins (targeting DsRed2) into rice zygotes in transgenic rice plants expressing DsRed2. DsRed2 expression is reduced in zygotes and/or subsequent embryos when sgRNA in ribo- nucleoproteins targets the DsRed2 sequence. As the method uses plant zygotes it does not require selectable marker gene to edit plant genome. Zygotes with decreased DsRed2 signal regenerate in to mature plant in the absence of any selection agents, with 14–64% showing targeted mutations. This study highlights the enormous potential of this method in other important crop too such as maize and wheat [95]. Dong et al. develop marker-free transgenic carotenoid rich rice using CRISPR/Cas9. The 5.2 kb marker-free carotenoid cas- sette is inserted at genomic safe harbour regions in the plant genome. Transformed dehusked seeds develop golden colour due to the presence of carotenoids in endosperm compared to wild type seeds [96]. Li et al. develop blight resistant transgenic free rice. In this study, partial sequence of Xa13 promoter was edited by CRISPR/Cas9. This does not alter the gene expression and function but alter its ability to get induced by pathogen. This editing improves the rice abil- ity to resist disease without affecting its fertility. PCR was used to identify the mutation site induced by double target sgRNA [97]. Conclusion Rice, after wheat, is the world’s second-largest source of human food energy. Innovative crop improvement technolo- gies are need of the hour to meet the growing food demand of the world’s population. This research focuses on advance- ments in rice transgenics for enhanced traits, with a focus on selection approaches that range from the use of existing SMGs to selectable marker-free progenies. Popular first gen- eration SMGs including hph, hpt, and nptII are regarded as threat to humanity if not removed before reaching the con- sumer. Second-generation selectable markers like as manA and xylA have not yet been discovered as possibly detrimen- tal to the environment, but they are still unappealing to con- sumers. As a result, transgene-free plants are the most attrac- tive and commercially acceptable. Since the first transgenic crop was released in 1996, there has been an 87-fold increase in the number of GM crops adopted around the world. Sev- eral ways in combination with the CRISPR/Cas9 editing tool, have been demonstrated as beneficial in rice genome editing. This have been achieved either by removing SMGs or by genome editing without the need of transgene integra- tion. However, no such GM crop has yet been sold, leaving plenty of room for experimentation. Similar rice engineering
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