Stress either abiotic or biotic adversely affects rice productivity. Enhanced yield potential may be achieved by employing suitable biotechnological approaches. Plant transformation technique is indispensable for the introduction of novel traits in crops for nutritional and yield improvements thereby ensuring global food and nutritional security. Recently, numerous findings with respect to in vitro regeneration and transformation approaches in umpteen rice genotypes have offered an ingenious platform for the nutritional and yield enhancements [25, 26]. However, predominantly indica rice cultivars around the globe, persist to be unpliable to genetic improvements owing to their exiguous regeneration potential. The existent protocols for regeneration and transformation of indica rice have emerged as arduous, protracted, and stringently genotype-specific with decumbent transformation efficiencies [21, 22]. The indica rice genotypes are invariably recalcitrant to tissue culture and callus induction & its regeneration involves utilization of genotype-dependent differential media combinations. Hence, implementing any one optimal hormonal-media therapy for triggering callus formation & its efficient regeneration across of indica rice genotypes is an enormously tedious task. The development of a genotype-free, in vitro regeneration & plant transformation technique provides a crucial platform for the improving crop breeding performances via rice genome modification employing the CRISPR/Cas9 approach. Taking into account the implication of plant transformation in translational genomics and crop genetic improvement, we devised a robust, reproducible, universally adaptable and inordinately dynamic transformation and regeneration protocol for various indica rice genotypes to overcome the unwarranted genotype-dependent optimization. We established a genotype-free, replicable method for biolistic-mediated transformation of indigenous indica rice (Oryza sativa L.) genotypes namely, Samba Mahsuri (SM) that is widely cultivated in southern belt of India comprising of 1-2 million hectares of Telangana, Andhra Pradesh, Tamil Nadu and Karnataka as well as Orissa, Bihar and UP, and White Getu (WG), as well as Hamilton (HT) with inherent salt tolerance, employing mature seed-derived calli. We earmarked the elite cultivar or mega-variety i.e. Samba Mahsuri (SM) (BPT 5204) for optimization of different parameters crucial for invoking improved transformation and regeneration frequencies. In order to devise a universal protocol for transformation and regeneration and for the revival of indica rice cultivars of Sundarbans, the standardized protocol was extrapolated to these salt tolerant genotypes WG and HT that catered to the agrarian community in the low-lying islands of Sundarbans, but were overnight lost to the tropical cyclone Aila that ravaged West Bengal in 2009.
Overview of an innovative genotype-free indica rice transformation protocol
Plant transformation entails few principle steps for instance, embryogenic calli generation, regeneration of shoots and induction of roots, however optimization of all these steps is requisite to suit individual plants. To develop a standard reproducible transformation protocol for rice (Fig. 1), we examined the effects of different combinations of growth regulators on callus- and shoot- regeneration by modifying existing protocol described by Kaul et al. [19]. Findings have been delineated under the following sections.
Induction of Embryogenic calli (EC) in mature seed explant
In order to obtain EC, we employed sterilized mature seeds as explants. First and foremost, step for a successful plant transformation technique is to obtain sterile or microbial contamination-free cultures. In the present investigation, explant disinfection was performed in three indica rice genotypes. A two-step sterilization process was employed to disinfect the seeds. Firstly, seeds were immersed in 70% ethanol for 2 min, followed by immersion in 2% sodium hypochlorite for 18 min and finally washed by sterile H2O (Additional file 1: Table S1). A low rate of seed contamination was observed in all varieties (SM=1%, WG=1%, WG=2%) after cultured on callus induction media. (Additional File1: Table S1).
Varying types of explants were employed for induction of EC for instance, leaf base; leaf sheath cells; root; immature- and mature seed-derived embryos. Amongst all, mature seed-derived embryos have been enormously utilized in rice in vitro culture studies [19, 27]. The efficacy of callogenesis and regeneration from explants tissues is influenced not only by the type of explants, physiological status of the explant genotype, but also by the suitable culture medium supplemented with different combinations of plant growth hormones [2]. Indica rice seeds mostly developed into non-EC with extremely low regeneration efficiencies and tedious than japonica subspecies as reported previously [5, 27-30]. Hence, to attain enhanced regeneration efficiency in indica rice we utilized mature seed-derived embryos for development of EC from indica rice genotypes (SM, WG, and HT) due to their easy accessibility throughout the year. To analyze the effect of supplementing an auxin & cytokinin amalgamation on callus induction (CI), eight different media combinations were designed (Additional file 1: Table S2). Sterilized seeds of the three genotypes were inoculated in two different basal media [Murashige & Skoog (MS) and Chu N6] containing differential combinations of growth regulators, i.e., 2, 4-D, dicamba, TDZ, glutamine, and other additives. We observed indistinguishably enhanced callus induction frequencies (CIF) when mature seeds of SM, WG & HT were placed on the two different basal media. Calli initiated invariably from the scutellar region of the embryo and a visible mass was generated within 5-d. Subsequently, the 15-d-old calli were sub-cultured for another 10-d period to obtain compact, nodular, light yellow or off-white coloured EC of 5-7 mm in size (Fig. 2a-b and 3; Additional file 2: Fig. S1: a-d). Significant differences were observed in CIF (%) when mature seed were cultured on MS- and N6- media supplemented with different combinations of growth regulators. It was revealed that on media containing 2,4-D alone, the percentage of EC was lower (60-75%) than that generated on 2,4-D with other additives (96-98%) (Table 1). We observed efficient CIF in all three genotypes on MS (96-98%) [CIMM7; Fig. 4a; Table 1] and N6 (95%) [CIMN7; Fig. 4b; Table 2] media when supplemented with 2, 4-D (2.5 mg/L); dicamba (1.5 mg/L); TDZ (0.1 mg/L); proline (1000 mg/L); and glutamine (2.5 mg/L). As the MS-based medium supplemented with growth hormones revealed a higher CIF (%) than N6-supplemented medium for EC formation (Additional file 1: Table S3), we chose the MS supplemented media for further experiments. Further, the 25-d-old calli generated on both CIMM7 & CIMN7 media showed enhanced proliferation and higher fresh weight (Fig. 4c) than those induced on other CI media (Table 3). These finding reveals that presence of 2,4-D (2.5 mg/L) with other additives is conducive for induction of embryogenic callus. In addition, increasing 2,4-D concentrations beyond 2.5 mg/L reduces the frequency of CI. During our experimentation, we found that on employing maltose (3%) as a carbon source, in place of sucrose effectuates higher EC induction. Osmotic status of the callus may be controlled via supplementation of basal media with maltose, which leads to higher induction rates of EC. Amino acids, for instance, proline & glutamine as media additives proved efficacious for the initiation and sustenance of EC. Moreover, inclusion of thidiazuron (TDZ), dicamba, and casein hydrolysate in the culture media led to enhanced somatic embryogenesis. In view of our findings, we recommend CIMM7 (MS- or N6-based media) for genotype-free embryogenic callus induction with no significant variations in the CI responses and confers enhanced TE and shoot RF in the selected indica genotypes. Furthermore, this media composition may overcome the much-hyped consequential variations in CI & regeneration frequencies prevalent amongst the rice genotypes. Henceforth, our proposed CI composition is a panacea or a consensus common medium, which ensures induction of EC in different Indica rice genotypes, a feat previously far-fetched. Thus, it may be inferred that CI is solely dependent on the permutation and combination of the growth hormones employed to supplement the basal media and independent of the genetic potential of the selected genotype. Overall, the statistical significance analysis of the above-mentioned dataset employing one way ANOVA (p ⩽ 0.05) followed by Tukey HSD (HSD0.5) tests demonstrated that CIF efficiency is influenced by the culture medium compositions.
In vitro regeneration with partial desiccation
The availability of an effective regeneration protocol is quintessential for plant transformation. Present investigation aimed to develop Cas9 transgenic rice lines via incorporation of a gRNA-free CRISPR/Cas9 vector employing biolistic-mediated transformation. This may serve as a platform for rice genome editing in the presence of the desired gRNA/s. To achieve successful biolistic transformation, a compatible robust reproducible regeneration protocol was developed prior to transformation. It is well known that plant morphogenesis and growth is maintained by proper auxin and cytokinin ratios. In the course of our investigation, we established that diverse growth regulators and their dosage employed in the regeneration medium play a crucial role in modulating the regeneration frequency. In a bid to endow higher regeneration frequencies, we harmonized the ratio and proportion of the growth regulators systematically to effectuate optimal RFs in all the three indica rice cultivars. Hence, we optimized the medium conditions for in vitro shoot regeneration utilizing MS basal media supplemented with varying permutations and combinations of auxins and cytokinins. To achieve the aforesaid objective, we employed different media compositions (RGM 1 to 8; Refer to Additional file 1: Table S4) for regeneration of calli, wherein RGM6: BAP (1.5 mg/L); NAA (0.5 mg/L); TDZ (1.0 mg/L); zeatin (0.2 mg/L) and proline (500 mg/L) rendered the highest regeneration efficiency of the calli incubated on it i.e., SM (94%), WG (92%) and HT (90%), respectively (Fig. 5a, Table 4, and Additional file 1: Table S5). Shoot primordial initiated after 7 days of the inoculation of 25-d-old EC on the regeneration medium (Fig.2c; Additional File 2: Fig. S1-e), followed by elongation of shoots (Additional File 2: Fig. S1-f). Regenerated shoots that adequately elongated after four weeks (Fig.2d; Additional File 2: Fig. S1-g) were transferred to the rooting medium (Fig. 2e; Additional File 2: Fig. S1-h). Our results displayed that fortification of RM (RM1-5) with zeatin (0.2 mg/L) and proline (500 mg/L) resurrected into a unique combination of growth regulators (BAP, TDZ, zeatin and NAA) termed as RGM6, which conferred a phenomenal boost-up in the regeneration efficiencies of EC, thereby invoking vigorous emergence of multiple in vitro shoots. Hence, the synergistic impact of exogenous zeatin (0.2 mg/L) and proline (500 mg/L) in the regeneration media (RGM6) was instrumental in increasing the shoot regeneration potential from rice seed-derived calli via stimulated cell divisions.
In order to evaluate the effect of desiccation on the regeneration efficiency of non-transformed calli, the mature seed embryo-derived calli were subjected to two experimental conditions, i.e., with- (24 h, 48 h, 72 h) & without-desiccation and assayed for differential efficiencies of regeneration (Fig. 5b). Noteworthily, incorporation of the dehydration or desiccation stress step, promoted somatic embryogenesis & shoot regeneration frequencies. Amongst all the calli desiccation treatments, the 48h desiccation period enhanced regeneration frequency (RF) in the three rice varieties (SM=94%; WG=92%; HT=90%) in comparison to non-desiccated fresh calli (SM=84%; WG=82%; HT=80%) (Additional file 1: Table S6). Therefore, we concluded that the 25-d-old calli, when subjected to 48h partial desiccation phase exhibited maximal regeneration frequencies (90-94%). The ANOVA (p ⩽ 0.05) followed by Tukey HSD test (HSD0.5) test results showed that regeneration efficiency and plantlet regeneration were mostly influences by the media compositions sans genetic potential of the genotypes. Thus, the RGM6 in consonance with partial desiccation of calli proved to be most efficacious in significantly enhancing regeneration efficiencies. Hence, it may be inferred that besides CI, regeneration efficiencies of EC are predominantly dependent on the permutation and combination of the growth hormones employed to supplement the basal media and independent of the genetic potential of the selected genotype.
Biolistic transformation and rhizogenesis of transformants
In vitro regeneration-based plant transformation approaches offer a crucial platform for basic and translational studies in plant science. Partially desiccated mature seed derived EC were the most preferable explants for biolistic transformation due to their higher RF. For efficacious delivery of CRISPR-Cas9 reagents, we employed an optimized biolistic-mediated transformation approach. Umpteen factors affecting bombardment efficacy were optimized to establish a simple and reproducible technique of transformation in indica genotypes (SM, WG, HT), utilizing the indigenously developed NICTK-1_pCRISPR-Cas9 marker-free vector that harboured the cassette of codon-optimized Cas9 gene (Additional file 1: Table S7). Different factors were optimized for instances, particle dimension (0.4-1.0 µm), target distance (3.0-9.0 cm), helium pressure (1,100 psi), and concentration of DNA (1.5-2.5 µg/shot). When EC were shot with gold particles (sized 0.6 µm) carrying 2µg/shot of DNA, employing a helium pressure of 1.100 psi at an appropriate target distance (9 cm) generated the highest transformation efficiencies (TE) in indica genotypes (SM, WG, HT) (Fig. 6k; Additional file 1: Table S7).
In addition, significant enhancements in regeneration efficiencies of bombarded calli were observed when pre-incubated onto CI media with variable osmotica for instance, mannitol, sorbitol and AgNO3. Calli placed on CI media supplemented with mannitol (36g/L), sorbitol (36g/L), & AgNO3 (5mg/L)-a potent inhibitor of ethylene action for 48h prior to bombardment, invigorated regeneration efficiencies (SM=88%; WG=87%; HT=84%) than those cultured on regeneration media sans supplementation of osmoticum (SM=84%; WG=82%; HT=80%). Regeneration efficiencies of transformed calli were lower (81-86%) than non-transformed ones (90-94%) (Fig 6j; Additional file 1: Table S8). The transformed calli were kept in dark for another 7-d-period on CI media and then transferred to regeneration media (RM) for induction of in vitro plantlets (previously described in CIMM7 & RGM6). The plantlets were eventually transferred to a hormone-free MS (half strength) medium for rhizogenesis (Fig. 2e; Additional File 2: Fig. S1-h). Consequently, a high frequency (100%) of root proliferation was recorded in the three varieties after 7-10 days of transfer. Shoots that attained a height of 3-4 inches and developed adequate roots were hardened in small pots with soil for a week (Additional File 2: Fig. S1-i). The hardened seedlings were further transferred to greenhouse field maintained at controlled temperature and humidity (Fig. 2f; Additional File 2: Fig. S1-j). Globally, numerous plant transformation strategies for instance agrobacterium- and biolistic-mediated methods, nanoparticle- and viral vector-based deliveries have been employed to incorporate useful traits for both indica and japonica genotypes [19, 24, 31]. Interestingly, sufficient reports highlight the efficient recovery of transgenic indica rice plants from calli utilizing the biolistic approach. Remarkably, our protocol emerges as a crucial intervention in elevation of the indica rice transformation efficiencies to a great extent (65-69%) than previously reported figures (47%) [32].
Molecular screening and validation of transform plantlets
In recent past, numerous reports have demonstrated the differential applications of CRISPR/Cas- based genome editing in plants. The genome edited plants have been interrogated employing multiple screening techniques to validate the indels or mutations, thereby incorporating the desired traits [7, 8, 9, 11, 12, 22, 26]. We elucidate the development of transgenic plants in recalcitrant indica rice genotypes via transferring the CRISPR/Cas9 reagents into rice genome employing biolistic approach. In order to validate if exogenous Cas9 gene was integrated into the rice genome, genomic PCR analysis of the transgene in transgenic lines was performed. Genomic DNA amplification of a Cas9 gene fragment sized 531bp was observed in the putatively transformed T0 plants in comparison to wild-type (WT) (Fig. 6a-c; Additional File 2: Fig. S2:a). PCR analyses of transgenic lines revealed that the highest transformation efficiency was achieved in SM (69%) in comparison to WG (67%), and HT (65%) (Additional file 1: Table S9). Incidentally, the TE reported previously in SM was recorded as low as 47% employing agro-mediated transformation by Reddy et al. [32]. Contrastingly, our protocol highlights a significant enhancement in transformation efficiency (69%) in this SM indica rice genotype. Moreover, a minimal TE of 6.5% was obtained via the particle-bombardment approach reported by Cho et al. [33]. Notably, this is the first report to establish a robust regeneration, and transformation protocol for salt tolerant varieties, i.e., White Getu and Hamilton. Indeed our ingenious protocol offers a robust, suitably optimized, genotype-free, universally applicable transformation strategy that may be effectively extrapolated to any rice cultivar, globally.
Subsequently, we performed digoxygenin (DIG)-based Southern blot analysis employing a Cas9-specific probe to ascertain the number of transgene integration sites for the PCR positive lines (Fig. 6g-i; Additional File 2: Fig. S2-b). The gDNAs of selected PCR positive transgenic plants from each genotype and their respective WT plants were digested with the restriction enzyme EcoRV as a single restriction site occurred in Cas9 gene. After hybridization with a non-radioactively labeled Cas9 gene-specific probe followed by autoradiography, varying sizes of hybridization signals or bands that corresponded to Cas9 gene fragments were observed (Fig. 6g-i; Additional File 2: Fig. S2-b). Most of the plants revealed a single copy of Cas9 gene insertion and two out of 18 transgenic lines revealed one to two copy insertions, thereby demonstrating stable transgene integration into the rice genomes (SM, WT, HT). On the contrary, no bands or signals were detected in WT lanes. Southern blot signals substantiate the Cas9 gene copy number in the transgenic plants as independent events. Based on the Southern analysis, we inferred that the PCR positive transgenic lines were independent transgenic events. Further, the PCR and Southern positive lines from each genotype were validated via Sanger’s sequencing (Fig. 6d-f; Additional File 3: Fig. S1-S6). Selected transgenic T0 plants were selfed, and seeds of individual lines were collected and subsequently germinated upon MS (half-strength) medium for further analysis. Upon germination, the T1 seedlings were PCR analyzed for the presence of Cas9 transgene employing gene specific primers. Results showed that the targeted Cas9 gene was inherited with a 3:1 Mendelian ratio for single-copy insertions (Additional file 1: Table S10). Hence, results suggested that Cas9 transgene was stably delivered to subsequent generations.
Phenotype of transgenic rice lines
In a bid to determine if the presence of Cas9 transgene would affect the morphology of the transgenic plants, we monitored their phenotypes. Mature seed-derived in vitro WT and transgenic T1 plants of the three genotypes were allowed to grow in field under controlled temperature and humidity conditions, to be sampled randomly. Their morpho-agronomic trait performances, for instance, plant height, leaves length & width, tiller numbers, number of productive panicles, length of panicles, filled grains/spike, 1000 grains weight, and so on were examined. We observed that in vitro regenerated WT and transformed plants were phenotypically indistinguishable and revealed better performances than WT (Additional file 1: Table S11). The Cas9 transgenic rice lines did not display any morphological variations in comparison to the WT (Table 5 and Additional file 2: Fig. S3-S5). In addition, the enhanced number of filled grains or spikes were obtained in SM, WG & HT transgenic lines, which revealed sustainably good agronomic performances (Additional file 1: Table S12). Moreover, the weight of 1000 grains revealed in the order of highest to lowest in the three genotypes was SM>WG>HT (Fig. 7). Furthermore, at maturity, all the confirmed transgenic rice lines were fertile and exhibited normal phenotype. Subsequently, we recorded the agronomic trait performances for transgenic and WT plants under simulated field conditions and astoundingly the transgenic lines of SM, WG, HT showed high yield potentials (Fig.7). Taken together, results strongly suggested that SM, WG & HT varieties responded efficaciously to the devised plant transformation protocol that may be utilized for further improvement of their nutritional and yield potentials via CRISPR/Cas9-based gene editing approaches.
In addition, we notably attribute the robustness of our method to few critical factors; for instance, highly efficient mature seed explant; improvement in CIF by addition of maltose (carbon source), proline & glutamine (amino acids), enhancement in RF with partial desiccation approach; biolistic-mediated DNA delivery approach, post-bombardment recovery on regeneration media and transgenic rice lines displaying efficient integration of Cas9 gene into the rice genome. The protocol presented here has significant advantages over the methods currently available especially in recalcitrant indica rice cultivars, for instance, higher CIF, large number of regenerated transgenic plants per calli within a short time span, stable transfer of transgene copy in subsequent generation. This novel protocol (Fig.1) offers an efficacious, viable, genotype-free strategy for genome editing in indica rice, in order to introduce agronomically important traits. Undoubtedly, our innovative protocol offers a robust platform that may be universally adaptable transformation strategy rapidly extrapolated to all rice cultivars, globally.