Genetic engineering refers to the modification of an organism’s genome. The modification may consist of a change, deletion or addition of a nucleotide base pair. It may, additionally, also include a transfer of gene segment from another organism into the host cell. Genetic transformation specifically refers to the techniques that are employed to obtain organisms that have been modified genetically, called the GMOs (genetically modified organisms). G. hirsutum is one of the first major crops whose transgenic varieties were introduced into the market globally. The crop finds basic application in consumer and industrial products such as in the textile industry. Cottonseed oil contributes to the food industry while seedcake is the major protein source in demand in the feed industry. Cotton exports generate billions in profit. Generally, the transformation of elite cultivars is preferred because they are adapted in farmers’ fields for agronomic characters. Monsanto’s Bollgard-I and Bollgard-II contained single and stacked Cry genes which were obtained from Bacillus thuringenesis that releases δ-endotoxins rendering the cultivars tolerant to pests (Qaim 2010). Similarly, competing weeds were eliminated by the use of herbicide-tolerant cotton cultivars (containing the cp4 EPSPS gene), such as Roundup Ready Flex (RF) cotton which was the first glyphosate-tolerant cotton (Puspito et al. 2015). Currently, various transformation methods have been employed to genetically-modified cotton. These methods include Agrobacterium-mediated transformation, protoplast treatment with PEG, particle bombardment, pollen tube pathway, and electroporation. Genetic engineering provides a promising alternative to genes that can otherwise be obtained from crop wild relatives or even unrelated sources like bacteria, fungi, animals, or plants.
Due to limited germplasm and the use of the same genetic sources, the conventional breeding methods no longer seem to be as promising to bring major uplift in cotton production. The development of stable transformation methods has paved the way for the improvement of the cotton genome. Since the development of the first transgenic cotton plant, back in 1987 many traits related to biotic and abiotic stress tolerance, and increase in cottonseed oil, fiber quality, and production have been explored. Thus, merging conventional breeding methods with genetic engineering contribute to improved yield and quality of cotton. The adoption of Bt cotton has changed the traditional breeding patterns including the use of agrochemicals with an observed improvement in the quality and quantity of the produce. The contribution of Bt cotton needs to be analyzed in detail considering global cotton cultivation. Various factors such as climate conditions, lack of quality germplasm, use of agrochemicals, and irrigated areas have forced the extensive adoption of Bt cotton by farmers around the globe. Thus, it is imperative to investigate the benefits of genetic engineering and its prospects (Bakhsh et al. 2015; Noman et al. 2016).
There is room for the improvement of abiotic and biotic stress tolerance since related determinants are unavailable within a crossable gene pool. Thus, employing genetic engineering seems inevitable for stable gene transformation. The last two decades resulted in more than 80% of global transgenic cotton (Juturu et al. 2015). Despite the huge success of transgenic cotton, various combinations of stresses keep on harming in-field cotton owing to the ever-changing environmental and climatic aspects. Resurgences of diseases are observed in recent times such as the appearance of bacterial blight and Fusarium wilt in the U. S. in 2017 that lead to a $45 million in yield loss (Cox et al. 2019).
Agrobacterium-mediated transformation refers to the genome transformation by utilizing the capability of the Agrobacterium to transfer foreign genes into plant host cells. It makes use of its Ti plasmid whereby the T-DNA is integrated into the host plant genome such that it is transferred to the offspring (Li et al. 2017b). A basic transformation protocol includes the application of a slight injury to plant tissue. This site is needed for exposure to the Agrobacterium carrying the desired gene construct. Transformation frequencies have been improved following several tested protocols including the use of super-binary vectors, vir gene inducers, ternary system, and modifications of the Ori of the vectors amongst others. A binary vector consisting of additional virulence genes from a Ti plasmid to enhance the transformation frequency is refered to as a super-binary vector (Komori and Komari 2011). Vir gene inducers refer to factors that facilitate the expression of vir genes which include, amongst others, acetosyringone, vanillin caffeic acid (Simon et al. 2015). A ternary system makes use of an accessory plasmid that is a virulence helper plasmid to help carry additional virulence gene cluster. It is essentially a three accessory system consisting of disarmed Ti plasmid, a helper plasmid and an additional virulence helper plasmid (Anand et al. 2018). A broad host range origin of replication ensures a high plasmid copy number resulting in an increased T-DNA transfer. For instance, the pSa plasmid produce 2~4 copies per cell, RK2 and pVS1 plasmids are reported to maintain 3~12 copies per cell while 15~20 copies per cell are obtained by the repABC origin (Zhi et al. 2015; Vaghchhipawala et al. 2018).
The choice of Agrobacterium strains needs to be considered based on the choice of crop. Cho et al. (2014) reported AGL1 to be the most effective strain in maize in comparison to LBA4404, EHA105, and GV3101. Alternatively, for effective transformation in cotton, the use of EHA105 and LBA4404 strains seem promising (Zhang et al. 2014). Transformation frequencies of 33% by strain AGL1 and 10% by strain LBA4404 were observed in sorghum (Wu et al. 2014).
Important plant tissue components used in optimized combinations help increase transformation efficiency even in recalcitrant crops. For instance, Cho et al. (2014) were able to establish optimum combinations of glucose, cytokinin, and copper for co-cultivation, resting and selective media in maize. In addition, the choice of tissue to be transformed should also be taken in consideration. Meristem cells are preferred since the foreign gene can be easily accepted by these cells upon quick division. Such cells also tend to be less probable to have somaclonal variations and genetic mutations (Kalbande and Patil 2016). According to Rajasekaran (2019a), a variety of explants may be transformed through particle bombardment. For example, transformation of apical meristem would ultimately help in deriving transformed shoots, bypassing regeneration steps, and thus reduce the time to obtain adult plants. In an experiment demonstrating particle bombardment, Rajasekaran (2019b) bombarded gold particles coated with β-glucuronidase or uidA on isolated mature seeds. The L1 layer was observed to be stably transformed while a reduced transformation of germ line was obtained, i.e., up to 0.71%.
Many transformation processes are aided by the co-culture medium of the explants. Transformation frequency (TF) can be enhanced at this stage in the presence of a certain optimized temperature. Cotton meristem transformation was reported by Chen et al. (2014) to achieve the highest efficiency at 23 °C and even a slight increase in degree would reduce the transformation levels. On the contrary, the transformation of cotton embryogenic callus was most favorable at 19 °C. This indicates that different explants may require different temperatures for effective transgene integration. Transformation may also be influenced by the time of Agrobatcerium inoculation. The pistil drip inoculation was carried out by Chen et al. (2010), with the suspension of Agrobacterium in a 10% sucrose inoculation solution containing Silwet L-77 and acetosyringone for the transformation of bar gene. Inoculation in the evening yielded ~ 0.07% to 0.17% herbicide resistant plants. This efficiency was further increased to 0.46%~0.93% upon excision of stigma prior to inoculation. On the contrary, a low rate of transformation (0.04%~0.06%) was observed when inoculation was carried out in the morning. In stark contrast, to aforementioned example, morning inoculations were preferred by Mogali et al. (2013). Treatment of stigmatic surface by 5% sucrose solution amplified the success of pollination up to 23.5%. Additionally, rate of boll set was observed to marginally increase with the use of boron. Hence, a combined application of sucrose and boric acid yielded a 32.5% increase in boll set. Boll shedding is a definitive occurrence following Agrobacterium application. Though, a liquid agroculture resulted in less boll shedding than a solid agroculture (making use of agar medium) did.
In addition to the various mentioned factors, the choice of transformation methods also influence the success of transformation. In case of biolistic method of transformation, some of the factors upon which the success of bombardment depend include the pressure applied, the choice and distance of the tissues along with the metal used for the particles. Kharbikar et al. (2013) used embryonic axes of cotton cv. NH 545 for the transformation of Cry1Ac gene pBin Bt-3. Bombardment of gold particles at 900 pounds per square inch (psi) at 6 and 9 cm yielded an overall gene transfer efficiency of 3%. On the contrary, the use of explant by Khan et al. (2011) was the excised cotton embryos. Tungsten was used as the materials for micro-projectiles with a pre-optimized distance of 22 cm at a pressure of 4.13 bar. A transformation efficiency of 0.26% was obtained. A critical observation of both the described experiments revealed a huge difference in the pressure applied. A 4.13 bar pressure is around 60 psi which is quite low compared with the 900 psi applied by Kharbikar et al. (2013). Additionally, explants at 6 and 9 cm are expected to have a higher transformation probability (as observed by the results) than those at a greater distance of 22 cm.
On the contrary, in planta transformation of plants usually evades the laborious and often recalcitrant regeneration procedures experienced in in vitro plant transformation techniques. Some methods that came under umbrella of in-planta transformation included injecting plant tissues with Agrobacterium, vacuum infiltration, floral dip, spray, and pollen-tube pathway (Niazian et al. 2017). The pollen tube pathway, for instance, essentially delivers the transgene into cotton embryo sacs for integration into cotton genome. Despite the apparent virtues, the transformation efficiency is low, i.e., about 0.5%~1.0% for majority transformations (Wang et al. 2013). Thus, in comparison to in vitro techniques, this method is independent on rigorous field bioassays before molecular characterization. Unlike co-cultivation that requires the application of an injury, the pollen tube pathway is pre-dominantly non-injurious, however, an absence of skilled handling and injection of increased volumes of DNA may damage the ovary. Moreover, the success of this technique is highly dependent on flower morphology (Ali et al. 2015). This is precisely why this method is not found to be favorable in soybean due to its small flowers with narrow and fragile staminal columns. Cotton, however, has large flowers can be easily pollinated.
A combination of microinjection and 20 s of sonication was reported by Gurusaravanan et al. (2020), to obtain high transformation efficiency in G. hirsutum L. KC3. Shoot apex explants of cotton were microinjected carefully to prevent damage followed by incubation for 1d in dark. On the next day, the explants were shifted to culture of Agrobacterium for sonication. The optimum shock time was 20 s beyond which the explant damage was increased greatly. Experiment was further optimized for best results with Agrobacterium cell density of 0.6 OD600 nm and three microinjections. Maximum transformation efficiency was observed to be 20.25%.
A variation in the gene expression levels is observed for different genotypes despite following the same transformation under similar conditions. Lei et al. (2012) reported the integration of SNC1 gene in Juanmian No. 1 and Zhong 35 cotton cultivars for resistance against Fusarium wilt. The disease incidence rates for Juanmian No. 1 controls and transformants were 66.7% and 37.5%, respectively, while for Zhong 35 controls and transformants were 50.0% and 22.2%, respectively. Such an observed variation in gene expression levels may be linked to a variation in the ease of transformation for both varieties.
To fully reap the benefits of transformation in cotton, it is incumbent to develop a cotton regeneration method whereby time can be saved. According to Bouchabke-Coussa et al. (2013), marker-free transgenic plants can be obtained with the use of Agrobacterium binary vector carrying WUS and the desired gene. It was observed that in vitro overexpression of AtWUS was synonymous with improved somatic embryogenesis and induced organogenesis on embryo-like structures in the absence of phytohormones. An increased fraction of explants leading to embryogenic tissues were obtained with the AtWUS-GFP (green fluorescent protein) which is probably due to the stability of the fusion product.
Various sugars and phenolic compounds are believed to induce vir genes for transformation. Acetosyringone is a prominent phenolic compound that contributes greatly to the effective transformation of Agrobacterium. Synergistic actions of various other compounds responsible for transformation may include vanillin, vanillic acid, caffeic acid, gallic acid, and coumarin as in the case of the microalga Dunaliella salina (Simon et al. 2015). Additionally, a down-regulation of o-methyltransferases has been reported to reduce the susceptibility of plants to agro-infection by lowering the virulence of Agrobacterium (Maury et al. 2010). The increased production of phenolic compounds may occur in vitro especially in the presence of certain C sources for certain plant species. For example, the presence of phenolic compounds is observable in cotton explants and is quite vivid in media containing sucrose. This is reported to hinder plant regeneration resulting in a reduced transformation frequency (Chen et al. 2014).
Instances of T-DNA transformation may further be increased with the removal of entities that may affect the plant-Agrobacterium interaction. This includes the removal of the gaseous phytohormone namely ethylene. This may be carried out by providing 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity to Agrobacterium which helps in cleaving ACC (the ethylene precursor) to ammonia and α-ketobutyrate (Nonaka and Ezura 2014). In a comparison study (Someya et al. 2013), ACC deaminase gene driven by virD1 promoter showed an increased ACC deaminase activity than with the use of lac promoter. Further, salicylic acid is also known to suppress the transcription of repABC operon, vir genes, and genes related to quorum sensing thus impeding the ability of Agrobacterium to infect plants (Someya et al. 2013). Agrobacterium-plant interactions are also affected by gamma-aminobutyric acid (GABA) which occurs by degradation of quorum sensing signals leading to a reduction in horizontal gene transfer of Ti plasmid. A combination of the ACC deaminase and GABA transaminase activity yields a super-Agrobacterium ver. 4 that is known to exhibit increased transformation efficiency (Nonaka et al. 2019).
More work needs to be done on visual marker system to aid genetic engineering and breeding in cotton. For example, Agrobacterium-mediated transformation was used to introduce red fluorescent protein, DsRed2 (obtained from Discosoma sp.), to be expressed in two cultivars, JIN668 and YZ1. An early-stage selection tool for transgenic calli is provided by DsRed2 which can be visually observed in calli, somatic embryos, and various tissues and organs in mature plants. In order to analyze its stable heritability, Sun et al. (2018) crossed the Yz-2-DsRed2 transgenic line with different cultivars. The progeny from a male parent showed stable inheritance with 100% expression in F1 hybrids. A negative association was obtained between DNA methylation and DsRed2 transcription following a methylation-specific polymerase chain reaction
(PCR) approach on CaMV35S promoter. Thus, DsRed2 was claimed to be a reporter gene for transformation and molecular breeding programs in cotton (Sun et al. 2018). Based on the increased sensitivity of upland cotton towards hygromycin, Bibi et al. (2013) claimed that hygromycin resistance should be the preferred choice for a marker over kanamycin resistance to screen mutant plants following a pollen tube mediated transformation.
Despite successful transformation events, the effect of transgenic crops may not have a pronounced effect in field. This discrepancy in the gene expression levels can be understood through spatio temporal studies of the crop. An in-depth explanation was provided by Bakhsh et al. (2012) whereby Cry2A toxin levels seem to fluctuate amid the crop growth duration. Additionally, different crop parts exhibit difference in toxicity levels, with the leaves having high toxicity than the reproductive parts. Moreover, the transgenic lines were observed to have a 100% mortality rate at 30 days age of plant which dropped to 60%~80% mortality by 90 days of crop age. Other factors that may influence transformation efficiency include the gene insertion point and its ultimate effect on the translation, inner environment of the cell based on the alterations in the outer environment and transgene copy number to name a few (Rao et al. 2011).
Interestingly, in vitro salt stress was observed to increase transformation efficiency which was demonstrated by Barpete et al. (2016). Salt stress was applied to 2-day old germinated embryos—50 mmol·L–1 of NaCl, CaCl2, KCL each which was followed by exposure to LBA4404 strain via co-cultivation. The highest transformation efficiency of 1.10% was achieved in embryos pre-treated with KCL, followed by NaCl and CaCl2 both at 0.7%. The results show a pronounced effect on transformation when compared with an efficiency in embryos not pre-treated with salts that stood at 0.4%.
Lee et al. (2013) reported the development of plant-transformation-competent binary bacterial artificial chromosome (BIBAC) library along with comparative genome sequence analysis of upland cotton with its putative progenitor’ G. raimondii. Both Agrobacterium and particle bombardment can be employed for the transformation of high molecular weight DNA vector. Digestion of DNA was carried out by BamH1 in pCLD04541. About 76 800 clones, with an average size of 135 kb, were present in the library having a probability of obtaining at least one positive clone with a single-copy probe. The various genes contained in the library were related to fiber cellulose biosynthesis and its development, cotton-nematode interaction, seed fatty acid metabolism, and resistance to bacterial blight. Randomly about 10 000 BIBAC ends (BESs) were selected for sequencing to understand the relationship of the upland cotton genome with G. raimondii. A major constituent of transposable elements was retro-element Gypsy/DIRS1 family in upland cotton. Both cultivars are greatly diverse at the genomic level.
Though transformation through in vitro techniques is deemed to be quite efficient, however, considering the reported transformation frequencies, this belief is debatable. The ease of the use of the various transformation methods are highly subjective depending on the optimized protocols and instrument availability in various labs around the world. For example, shoot apex transformation yielded 1.1% efficiency with PHYB gene upon 1 h co-cultivation with LBA4404 strain (Rao et al. 2011); a 1.19% efficiency with pyramiding of glyphosate resistance and Bt genes (Puspito et al. 2015); and 1.01% for the CpTIP1 gene (Akhtar et al. 2014). Decent instances of transformation are obtained through in planta methods. Some of the reported transformation frequencies following the cotyledonary leaf bisection method are 6.89% for At-NPR1 gene inducing resistance to Alternaria alternata (Kalbande and Patil 2016) and 2.27% for glyphosate tolerance up to 800~1500 mg·L–1 (Karthik et al. 2020). Manipulation of pollen tube pathway yields differing frequencies such as about 0.30% for induction of Helicoverpa armigera mortility (Mogali et al. 2013). Transformation through particle bombardment is believed to have slightly low frequencies and yield chimeras with more chances of epidermal transformants and co-suppression resulting due to multiple transgene copies with a greater probability of fragmented T-DNA (Chakravarthy et al. 2014). Reported transformation via particle bombardment include 0.71% efficiency with gold particles for β-glucuronidase (Rajasekaran 2019b), 3% efficiency with gold particles by Kharbikar et al. (2013) and 0.26% with tungsten particles by Khan et al. (2011). Despite the general belief, the transformation efficiencies are competitive.
Methods for confirmation of transformation
Verification of integration of desirable genes is important to the entire transformation process. Without this step the time and labor intensive techniques of genetic engineering are rendered inconsequential. The basic confirmation processes include molecular confirmation such as by Northern Blot Analysis, Southern Blot Analysis (Chen et al. 2010; Zhao et al. 2015; Zhang et al. 2014) in association with PCR carried out in thermal cycler (Sohrab et al. 2016). Variations of PCR may be employed as per the requirement of the experiment. These include reverse transcription PCR (rt-PCR) and real time PCR (q-PCR) (Maqbool et al. 2010; Zhang et al. 2014). Western blot can be carried out alongside enzyme-linked immunosorbent assay
ELISA (de Oliveira et al. 2016). The NCBI BLAST Pair-wise Alignment algorithm programs are helpful in sequence analysis (Maqbool et al. 2010). Additional assays include phenotypic expressions such as GUS (β-Glucuronidase) assay (Maqbool et al. 2010; Bibi et al. 2013). Insect bioassays determine the level of toxicity against unwanted pests and larvae (Mogali et al. 2013; de Oliveira et al. 2016). Initial selection parameters are usually limited such as to selection pressure antibiotics, herbicide, etc., including kanamycin, phosphinothricin (Mogali et al. 2013; de Oliveira et al. 2016). Robust molecular assisted selection (MAS) would ensure early selection amongst putative transformants. The selection is quick and prevents frequent field inoculations. A notable example includes the tight linkage of microsatellite markers with the disease resistance genes. Pyramiding of resistance genes in desirable cultivars is possible since the marker expression is not obscured by the epistatic interactions amongst resistance genes (Marangoni et al. 2013).
A modification of the PCR included the use of loop-mediated isothermal amplification (LAMP)—an essay that is highly sensitive, rapid and efficient. Amplification of the gene of interest was carried in the presence of loop primers that greatly reduced the time, otherwise, utilized by conventional PCR process. A rapid DNA extraction followed by LAMP assay is claimed to take about 30 min (Rostamkhani et al. 2011) and the obtained products can be visually observed in reaction tube upon observing the turbidity. Surface plasmon resonance (SPR) is another biomolecule based detection system that can easily identify transgenic cotton within 5 min per sample. Zhao et al. (2013) used this method to detect transgenic Cry1Ac cotton individuals. In this protein based analysis, a CM5 sensor chip served as a base to immobilize monoclonal Cry1Ac antibodies against conventional cotton samples that were used as the detection threshold. Fluorescent multiplexed immunoassays (FMI) have gained importance for stacked GM traits. Yeaman et al. (2016) developed an FMI assays for major transgenic proteins including neomycin phosphotransferase II, β-glucuronidase, CP4-EPSPS, Cry1Ac and Cry2Ab2. Characterization requirements and results for FMI are like ELISA but has reduced time with a higher throughput.
RNAi-mediated gene silencing
In October 2018, US regulators approved the TAM66274 event whereby “Ultra-low gossypol cottonseed” (ULGCS) plants were developed. Cottonseeds contain elevated amounts of terpenoid gossypol which is detrimental for human and livestock consumption. With the help of RNAi mediated silencing, δ-cadinene synthase was knocked down in the presence of α-globulin promoter, resulting in reduced levels of gossypol in cottonseed (Hagenbucher et al. 2019; Rathore et al. 2020). However, gossypol provides defense to plants against leaf-feeding pests suggesting a decrease in defense following its knockdown. Thus, the method was used to analyze the insect resistance of ULGCS cotton plants against Spodoptera littoralis which is a causative agent of African cotton leaf-worm. It is interesting to note that ULGCS have a significantly high oil content of about 4%~8% (Palle et al. 2013). Surprisingly, the stability has also reported to be multi-generational as far as five amongst nine RNAi lines against certain pathogens (Rathore et al. 2012), though the gossypol was naturally found in cotton and has deleterious effects on the cotton pests. Likewise, gossypol is dangerous for animals where it goes in seedcake. Keeping in view the adverse effects of gossypol, CYP6AE14 transcript was transformed in cotton with a construct of 469 nucleotide while being assumed that the enzyme detoxifies gossypol. This transcript specifically interacts with the cotton bollworm cytochrome P450 CYP6AE14 upon consuming the transgenic cotton leaves leading to reduced bollworm larvae population (Zhang et al. 2017).
Alternatively, an intron hairpin (ihp) RNAi construct was developed that was able to express dsRNA homologous to CLCuRV intergenic region (IR) (Khatoon et al. 2016). The Narasimha cultivar was transformed with Agrobacterium containing the construct yielding nine independent transformed lines. Resistance to CLCuD was observed after 90 days of inoculation with viruliferous whiteflies. RNA interference (RNAi) has also been regulated to develop cotton resistant to bollworms (H. armigera) larvae. The 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene plays a role in rate-limiting reaction of mevalonate pathway for juvenile hormone (JH) synthesis in cotton bollworm. This gene was allowed to be targeted by double-stranded RNAs (dsRNA). Feeding on leaves of transgenic cotton containing HMGR, its expression was significantly downregulated in larvae of H. armigera. The expression was as low as 80.68% compared with wild type. Additionally, an expression of vitellogenin was reduced up to 76.86% (Tian et al. 2015).
Silencing genes that code proteins essential for pests ensures safe and effective pest control strategy. RNAi can be used in combination with Bt toxins to achieve stronger durable pest resistance with an even reduced probability of pests becoming resistant (Tabashnik and Carrière 2017). Single Cry expression levels, at times, are low enough for the pests to survive the toxin. For example, Pectinophora gossypiella—a secondary cotton pest, is known to be tolerant to the low Cry toxin levels. Thus, a selected combination of traits can also delay the occurrence of resistance in transgenic cotton such as pyramiding of Bt toxin with RNAi, referred to as the next generation transgenic development. One such instance involved dsRNA production from Helicoverpa armigera to interfere its juvenile hormone synthesis by targeting JH acid methyltransferase (JHAMT) coupled with Bt toxin (Ni et al. 2017).
CRISPR/Cas system for cotton improvement
Plant breeders have successfully been able to develop high yield cotton with superior quality of lint. Seed yield and fiber length of fiber have been increased as a result of traditional breeding techniques. Additional yield gains were obtained by using improved cotton husbandry techniques along with the use of wild relatives for traits related to abiotic and/or biotic stress tolerance. Such improved accessions may be further used for genetic manipulation of latest techniques, e.g., CRISPR/Cas for improved traits by eliminating undesirable genes (Rauf et al. 2019). CRISPR/Cas system is renowned for its accuracy, ease and increased efficiency (Mao et al. 2013; Feng et al. 2013; Chen et al. 2017). Though, it has been demonstrated to be successful in various crops, CRISPR/Cas based cotton genome editing needs to be researched upon. Cotton carries significance for its fiber, oil and protein for feed industry. Targeting both sets of homologous alleles of tetraploid cotton was difficult, thus a transgenic cotton genotypes having a single copy of green fluorescent protein (GFP) was used to understand the efficacy of CRISPR/Cas9 system (Janga et al. 2017). Targeted mutations were confirmed upon losing GFP fluorescence and presence of various indels making use of three separate sgRNAs. Hence, CRISPR/Cas9 is deemed useful to target various genes within cotton genome. The efficiency of CRISPR/Cas system for allotetraploid cotton was also demonstrated by Li et al. (2017a), who detected 50% truncated events in transgenic individuals. Additionally, no off-target mutations were also detected.
Cytidine deaminase fused with CRISPR/nCas9 (Cas9 nickase) or dCass9 (deactivated Cas9) was proved to be effective for creating point mutations (Qin et al. 2020). To create single base mutations in cotton genome, a base-editing system for the G. hirsutum-Base Editor 3 (GhBE3) was developed. The CRISPR/Cas9 plasmid (pRGEB32-GhU6.7) was inserted with a cytidine deaminase sequence in fusion with nCas9 and uracil glycosylase inhibitor (UGI). For both target genes GhCLA and GhPEBP, three target sites were chosen for test accuracy and efficiency of GhBE3. The editing efficiency of three target sites lies in 26.67%∼57.78%. C-T substitution efficiency was found between − 17 bp and − 12 bp from protospacer adjacent motif (PAM) sequence. Additionally, no off-target mutations were found amidst 1 500 potential off-target sites in the genome. The T1 progeny was observed to inherit the edited bases.
To validate the functionality of sgRNAs to target three genes in cotton, Gao et al. (2017) analyzed the extent of CRISPR/Cas efficiency via a transient method. Mutations were generated in GhCLA1 and in GhPDS and GhEF1 at two target sites along with simultaneous editing of homeologous genes and deletions in polyploid cotton genome. Various sgRNAs expressing and targeting together indicated to highly efficient functionality with observation of 80.6% mutation frequency. A major composition of mutations included deletions of about 64% in cotyledonary tissues. The process is claimed to be accomplished in 3 days with the use of multiplex binary vector system (pYCLCRISPR/Cas9). Albino phenotypes were developed owing to targeting of GhCLA1 gene (Wang et al. 2017). It was intended to develop transgenic lines that can also be developed for breeding purposes using CRISPR/Cas 9 system. For instance, cotton arginase gene (GhARG) was knocked out from R18 which is a transgenic acceptor variety obtained from Coker-312. Successful events of gene knock out from A and D genomes resulted in improved lateral root development irrespective of nitric conditions.
A heat inducible CRISPR/Cas12b editing system was utilized to generate mutants with the highest editing efficiency by Wang et al. (2020). Hypocotyls were exposed to Agrobacterium for 2 days and then shifted to callus induction medium. The transgenic cells harboring the AacCas12b gene were exposed to 42 °C, 45 °C, and 48 °C for varying eight incubation times (that spanned to hours and days). All of the hypocotyls treated at 48 °C for more than 2 days failed to develop hinting at the in vitro limiting temperature. The plant exposed to 42 °C for 4 days exhibited simultaneous editing from two target sites. The highest editing rate was observed at 45 °C for 4 days along with the least damaging effects on the callus. Off-target mutations were absent. Heritability was observed to be stable in T1 generation.