Introduction

Radish (Raphanus sativus L., 2n = 18), associated with the Brassicaceae family (Gómez-Campo 1980), is an annual or biennial, commonly significant vegetable crop cultivated in tropical and temperate regions. It has been developed globally on 70,773 ha, predominantly cultivated in Japan, China, and Korea (Kurina et al. 2021). Each year, Japan produces 3.7 million tons of radish daikon and imports another 0.9 million tons from various countries. In contrast, China produced 1.2 million ha and occupied 6% of the cultivated area for vegetable crops (Kurina et al. 2021). It contains high levels of vitamin C (18%), potassium (5%), calcium (3%), iron (3%), and protein (1%) (USDA 2020). The significant edible portion of the juicy taproot in radish includes high nutritional content and health benefits (Yu et al. 2016; Pervitasari et al. 2022). Aside from the roots, the leaves and sprouts also have nutritious and therapeutic values (Takaya et al. 2003; Manivannan et al. 2019). Both round and extended radishes are consumed raw or pickled, preserved later, or boiled in various oriental cuisines (Park et al. 2005). It has a good source of antioxidants, including pyrogallol, vanillic acid, coumaric acid, catechin, and other phenolic compounds, which assist in preventing diabetes, neurological disorders, cancer, Parkinson’s, and cardiovascular diseases (Manivannan et al. 2019).

Conventional breeding is vital for designing superior radish varieties. Nevertheless, breeding success rates are hindered by sexual incompatibility and difficulty finding viable progenies (Elayaraja et al. 2019). Furthermore, developing a unique variety of breeding practices requires an extended period, which could be solved by genetic transformation strategies (Elayaraja et al. 2019). On the other hand, radish is recalcitrant to regeneration and demands a specific transformation strategy. Consequently, efficient regeneration and genetic transformation methods are necessary for essential vegetable crops like radish. The available regeneration and few reports of genetic transformation methods on radish have been observed employing hypocotyl and cotyledon explants that exhibit a low frequency of shoot regeneration, a limited range of plant growth regulators (PGRs), and less transformation efficiency (Paek et al. 1987; Pua et al. 1996; Curtis et al. 2004; Cho et al. 2008; Manawadu and Dahanayake 2015; Muto et al. 2021). These concerns would be solved by optimizing regeneration, including PGR conditions, and standardizing regeneration protocol to enhance transformation efficiency. A broad range of auxins and cytokinins have been previously used separately or combined with diverse vegetable crops (Elayaraja et al. 2019; Kapildev et al. 2020). Further, cytokinins recreated an essential role in shoot proliferation and were successfully adopted in radish (Jeong et al. 1995; Manawadu and Dahanayake 2015; Kozar et al. 2021). Consequently, mT is a natural aromatic cytokinin initially extracted from poplar leaves (Horgan et al. 1975) that plays an essential role in enhancing the growth of shoots and roots, increasing photosynthetic pigments, delaying senescence, and modulating the activities of antioxidant enzymes (Werbrouck et al. 1996; Aremu et al. 2012). In vitro mT regeneration has been successfully employed in several plants, including Aloe polyphylla (Bairu et al. 2007), Dierama erectum (Koetle et al. 2010), Merwilla plumbea (Baskaran et al. 2012), Drimia robusta (Baskaran et al. 2013), Sesamum indicum (Elayaraja et al. 2019), and Vigna mungo (Kapildev et al. 2020). To date, there is no report to describe the impact of mT on radish regeneration and genetic transformation efficiency. Hence, the present investigation aimed to understand the implications of mT with diverse PGRs for enhancing regeneration and genetic transformation efficiency in radish using the pCAMBIA1301 (Fig. 1) vector.

Figure 1.
figure 1

A linear map of the pCAMBIA1301 vector was used for transformation studies. CaMV 35S promoter-driven via hygromycin phosphotransferase II (hpt II) as a plant selection marker gene, whereas β-glucuronidase (gus A) as a reporter gene inside the T-DNA section.

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Materials and methods

Plant material

We have collected radish Pusa Chetki variety (var.) seeds (Fig. 2a) from Murugan Agricultural Distributors, Tiruchirappalli, Tamil Nadu, India, to regularize regeneration and transformation studies. This variety has been developed by the Indian Agricultural Research Institute (IARI), New Delhi, India, via selection from Denmark germplasm. It is remarkably tolerant of high temperatures. Roots are medium sized, mildly spicy, pure white, slim, soft with a blunt end, and harvested 40–45 d after planting.

Figure 2.
figure 2

Impact of mT on the regeneration of radish var. Pusa Chetki. (a) Mature radish seeds. (b) 7-d-old seedlings. (c) Cotyledonary node explant inoculated on SIM supplemented with BA (2.0 mg l−1) and mT (0.9 mg l−1) after 3 d of initial culture. (d) and (e) Shoot multiplication on SIM containing BA (2.0 mg l−1) and mT (0.9 mg l−1) after 3 and 3 wk of culture. (f) Elongated shoots on SEM including GA3 (2.0 mg l−1) and mT (0.9 mg l−1) after 4 wk of culture. (g) Rooted shoot on RM containing IBA (0.9 mg l−1) and mT (0.9 mg l−1) after 4 wk of culture. (h) Plants grown in a paper cup for 2 wk are maintained in a growth chamber. (i) After 2 wk, the plant hardened into greenhouse condition. Bars (a to i—1.0 cm).

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Surface sterilization, in vitro seed germination, and preparation of explants

Surface sterilization of radish seeds was performed as described by Balasubramanian et al. (2018). After sterilization, the seeds were shifted into a 150 ml Erlenmeyer flask comprising 40 ml of sterile distilled water and incubated at 25 ± 2 °C in a dark room for 1 d in a rotary shaker at 125 rpm. After 1 d, seeds were germinated on MS basal medium (Murashige and Skoog 1962) for 7 d at 27 ± 2 °C under a 16-h photoperiod. Seven-day-old seedlings (Fig. 2b) develop cotyledonary node explants (Fig. 2c) (~ 4 mm), which were collected by excluding hypocotyls, primary shoots, and cotyledons.

Optimization of PGRs and acclimatization

Cotyledonary node explants were inoculated on a shoot induction medium (SIM) containing various concentrations (1.0–3.0 mg l−1) of N6-benzyladenine (BA), kinetin (K), and zeatin riboside (Z). After 6 wk, the proliferated shoots were shifted into a shoot elongation medium (SEM) having different concentrations (1.0–3.0 mg l−1) of indole-3-acetic acid (IAA) and gibberellic acid (GA3). Subsequently, after 4 wk on SEM, they were sub-cultured for 4 wk on a rooting medium (RM) comprising various concentrations (0.3–1.5 mg l−1) of indole-3-butyric acid (IBA) and α-naphthalene acetic acid (NAA). The control explants were cultured on hormone-free MS basal medium for shoot induction, elongation, and rooting stages. All chemicals were procured from Hi-Media, Mumbai, India. All cultures were incubated at 27 ± 2 °C for 16 h with a photoperiod of 50 μmol m−2 s−1. The well-rooted plantlets were rinsed under running tap water and shifted into paper cups filled with a mixture of soil, sand, and vermiculite (1:1:1 v/v/v) and hardened for 2 wk in a growth chamber (Sanyo, Osaka, Japan) at 27 ± 2 °C, 60–80% relative humidity (RH). In the subsequent 2 wk, the plants were moved into earthen pots containing the above soil mixture and grown to maturity in greenhouse conditions. Furthermore, it would determine the optimized regeneration systems by employing mT.

Influence of mT on regeneration stages

We evaluated the impact of mT on regeneration stages for culturing explants on MS basal medium augmented with different concentrations (0–1.5 mg l−1) of mT (Hi-Media, Mumbai, India) along with optimum concentrations of SIM [MS, 3% sucrose, 2.0 mg l−1 BA, 0.8% agar (pH 5.6)], SEM [MS, 3% sucrose, 2.0 mg l−1 GA3, 0.8% agar (pH 5.6)], and RM [MS, 3% sucrose, 0.9 mg l−1 IBA, 0.8% agar (pH 5.6)] respectively. mT was filter sterilized using a 0.22-μm syringe-driven filter added after autoclaving in the medium noted overhead. The control explants were cultured on a separate medium comprising optimal PGRs without mT. All regenerated plants were hardened in similar conditions as described to optimize PGRs. Further, mT-supplemented radish regeneration utilizes transformation studies.

Sensitivity of the cotyledonary node and rooting explants to hygromycin B

We determined the sensitivity concentration of hygromycin B (Hi-Media) to the cotyledonary node. Initially, the explant was inoculated on SIM for 15 d devoid of hygromycin B. Explants were sub-cultured twice at 15 d intervals into fresh SIM, including various hygromycin B (0, 2, 4, 6, 8, and 10 mg l−1) concentrations for 30 d. After 45 d, explants were moved into SEM containing hygromycin B (0, 2, 4, 6, 8, and 10 mg l−1) concentrations for 30 d. Following 30 d, explants were transferred into RM supplemented with hygromycin B (0, 2, 4, 6, 8, and 10 mg l−1) concentrations for 30 d. Hygromycin B sensitivity concentration evaluates individual regeneration stages for lessening getaway. The control explant was grown in a hygromycin B-free medium. All explants were incubated at 27 ± 2 °C for a 16-h photoperiod at 50 μmol m−2 s−1.

Binary vector, Agrobacterium strain, and preparation of Agrobacterium suspension

The present investigation employed binary vector pCAMBIA1301 containing the Agrobacterium tumefaciens strain LB4404. The strain specifications and T-DNA portion of the binary vector pCAMBIA1301 were previously defined by Karthik et al. (2021). It has been cultured in Agrobacterium minimal (AB) agar medium comprising 10 mg l−1 rifampicin (Hi-Media) and 50 mg l−1 kanamycin (Hi-Media). A single colony was picked and inoculated with 10 ml of Luria–Bertani (LB) broth of seed culture, containing appropriate antibiotics as mentioned above, on a rotary shaker at 180 rpm, 28 °C for 48 h. From this seed culture, 300 μl was moved into 35 ml of LB broth with similar antibiotics and maintained at 180 rpm at 28 °C till it reached 0.8 at OD 600 nm. Bacterial cells were re-suspended in 35 ml of liquid infiltration medium [(LIM: MS, 3% sucrose, 3 mM 2-(N-morpholino ethanesulfonic acid) (MES), 2.0 mg l−1 BA (pH 5.4)] comprising 100 μM acetosyringone (AS) (Hi-Media) after centrifugation at 5000 rpm for 5 min. Subsequently, Agrobacterium suspensions were incubated at 28 °C at 130 rpm for 1 h before transforming.

Influence of mT on radish transformation

The cotyledonary node explants were gently pricked two times in the axillary and apical meristematic regions by a sterile hypodermic needle, 24-in. gauge (Hindustan Syringes & Medical Devices Ltd, New Delhi, India) and then transferred into the Agrobacterium suspensions for 30 min at room temperature with a rotary shaker at 90 rpm. Following infection, explants were dried on sterile tissue paper and shifted to co-cultivation medium [CCM: MS, 3% sucrose, 3 mM MES, 2.0 mg l−1 BA, 0.9 mg l−1 mT, 100 μM AS, 0.8% agar (pH 5.4)] for 3 d, maintained in the dark. After 3 d, explants were exhaustively washed twice with 350 mg l−1 cefotaxime, dried, and transferred to SIM containing 0.9 mg l−1 mT, 350 mg l−1 cefotaxime for 15 d, except for hygromycin B. After that, explants were moved into SIM with 0.9 mg l−1 mT, 350 mg l−1 cefotaxime, and 10 mg l−1 hygromycin B for 30 d. Following 45 d, explants were cultured on SEM with 0.9 mg l−1 mT, 350 mg l−1 cefotaxime, and 10 mg l−1 hygromycin B for 30 d. Finally, explants were inoculated with RM comprising 0.9 mg l−1 mT, 350 mg l−1 cefotaxime, and 6 mg l−1 hygromycin B for 30 d. All cultures were incubated at 27 ± 2 °C for 16 h with a photoperiod of 50 μmol m−2 s−1. Rooted plants were hardened in a similar condition as described to optimize PGRs. An individual study was performed without mT after infection in the respective medium mentioned overhead to explore transformation efficiency.

GUS assay and polymerase chain reaction (PCR) analysis

GUS expression in transformed and non-transformed (NT) explants was analyzed following Jefferson et al. (1987).Further, three randomly selected gus-positive 1-mo-old plants (anointed R1, R2, and R3) were chosen to verify hpt II (FP: 5′-ATGAAAAGCCTGAACTCACCGCGACG-3′; RP: 5′-CTATTTCTTGCCCTCGGACGAGTGCT-3′) and gus A (FP: 5′-ACTCGACGGCCTGTGGGCATTCAGTCTG-3′; RP: 5′-CACTGACCGGATGCCGACGCGAAG-3′) genes by PCR. The isolation of genomic DNA and PCR program strategies were followed as described by Karthik et al. (2018). Genomic DNA isolated from the radish NT plant and pCAMBIA1301 acted as negative and positive controls, respectively. PCR products were checked on a 1.0% agarose gel (w/v) (Hi-Media) and visualized under a Gel Documentation system (UVITEC, Cambridge, UK).

Reverse transcriptase-PCR (RT-PCR) and quantitative real-time PCR (qRT-PCR)

Total RNA was extracted from three hpt II gene PCR positive events of transformed and NT plants using the RNAqueousTM kit (Ambion Inc., Austin, TX). RT-PCR was executed per the kit instructions to operate one-step RT-PCR equipment (Qiagen, Germantown, MD). The hpt II gene was amplified from first-strand cDNA using the above primer, and ACTIN (FP: 5′-GCTTTTCCTTGATGTCTCTC-3′; RP: 5′-TCCTGCCATGTATGTTGCTA-3′) was employed as a reference gene (Pervitasari et al. 2022). Expected RT-PCR products were checked on a 1.0% agarose gel and visualized over a Gel Documentation System (UVITEC, Cambridge, UK). Furthermore, qRT-PCR was performed by the Light Cycler® 480 Real-time PCR system (Roche, Mannheim, Germany). Three hpt II gene RT-PCR positives and NT plants were examined by the Prime Script™ RT Reagent Kit (Takara Bio Inc., Shiga, Japan) to quantify the hpt II (FP: 5′-TATCCACGCCCTCCTACAT-3′; RP: 5′-CGACGTCTGTCGAGAAGTTT-3′) gene in triplicates. As per the kit recommendation, the reaction mixture is up to 50 μl, including water. The cycling parameters were described by Wang et al. (2019). Relative hpt II gene expression in transgenic radish was assessed by a cycle threshold (CT) value employing the 2−ΔΔCT method (Livak and Schmittgen 2001).

Statistical analysis

A hundred explants in triplicates were employed to statistically analyze regeneration and transformation strategies. Duncan’s multiple range test (DMRT) examined the data using one-way analysis of variance (ANOVA). At a 5% level, SPSS® 16.0 was used to calculate the significance level.

Results and discussion

Plant material

Cotyledonary node explants emanating from 7-d-old seedlings were employed in this investigation. Superficial mT has been revealed to significantly enhance regeneration in diverse crops (Baskaran et al. 2013; Elayaraja et al. 2019; Kapildev et al. 2020). Nevertheless, to date, there has been no report on the role of mT in enhancing plant genetic transformation efficiency. Consequently, the present investigation aimed to understand the impact of mT on improving direct regeneration and genetic transformation efficiency in radish.

Optimization of PGRs and influence of mT on regeneration stages

PGRs are an essential process for in vitro regeneration of plants. Explants cultured on SIM containing BA (2.0 mg l−1), SEM supplemented GA3 (2.0 mg l−1), and RM including IBA (0.9 mg l−1) showed the highest response in shoot multiplication (Table 1), elongation (Table 2), and rooting (Table 3) compared with other PGRs examined (data not shown). The control explants cultured on hormone-free MS basal medium exhibited the lowest response (data not shown). In vitro regeneration has been significantly enhanced, including mT (Elayaraja et al. 2019). In our examinations, a maximum number of shoots (32.6) was achieved when 0.9 mg l−1 mT was combined with BA 2.0 mg l−1 on SIM (Fig. 2d and e, Table 1). Kapildev et al. (2020) reported cotyledonary node explants cultured on SIM containing BA 0.5 mg l−1 and mT 1.5 mg l−1, resulting in maximum shoot regeneration in black gram. Elongated shoots (23.6) were highest on SEM containing 2.0 mg l−1 GA3 and 0.9 mg l−1 mT (Fig. 2f) as compared to SEM with GA3 alone (Table 2). Elayaraja et al. (2019) proved that adding mT to SEM substantially influenced the elongation of shoots in Sesamum indicum. Rooting (17.3) was highest when 0.9 mg l−1 mT was combined with 0.9 mg l−1 IBA on RM (Fig. 2g, Table 3). Similarly, mT has been successfully implemented in Corylus colurna (Gentile et al. 2017) and Actinidia chinensis (Saeiahagh et al. 2019) for root development. The well-rooted plantlet was hardened in a paper cup (Fig. 2h) in the growth chamber. After 2 wk, the hardened plants were shifted to an earthen pot and grown to maturity in the greenhouse (Fig. 2i), where 85% of the plants were established successfully.

Table 1. Impact of mT on shoot multiplication from cotyledonary node explants of radish on SIM after 6 wk of culture
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Table 2. Impact of mT on shoot elongation from cotyledonary node explants of radish on SEM after 4 wk of culture
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Table 3. Impact of mT on rooting from cotyledonary node explants of radish on RM after 4 wk of culture
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Sensitivity of the cotyledonary node and rooting explants to hygromycin B

A small portion of the target tissue or explant could be genetically transformed during the transformation strategy, and the remnant would be non-transformed. Hence, the selection agent should strictly recognize transformed cells and address them by employing a selection agent in a medium that includes hygromycin B. It is an antibiotic that interferes with protein synthesis by intercepting polypeptide elongation, and the demise of non-transformed tissue or plants assists in selecting transformed plants (Olhoft et al. 2003). We evaluated diverse hygromycin B concentrations and found that 10 mg l−1 hygromycin eventually hindered shoot induction and elongation from cotyledonary node explants, whereas 6 mg l−1 hygromycin B inhibited root growth from elongated shoots. Therefore, 10 and 6 mg l−1 (Fig. 3) hygromycin B concentrations were employed to select the transformed plants. Muto et al. (2021) revealed hypocotyl explants screened on SIM containing 10 mg l−1 hygromycin B in radish. Hence, hygromycin B serves as a promising selection agent that has successfully been introduced into diverse crops such as soybean (Arun et al. 2015), wheat (Zale et al. 2009), sorghum (Yellisetty et al. 2015), and bitter gourd (Karthik et al. 2021).

Figure 3.
figure 3

Sensitivity analysis of the cotyledonary node and rooting explants to hygromycin B.

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Influence of mT on radish transformation

Plant genetic transformation necessitates increasing regeneration and transformation efficiency (Karthik et al. 2020). Following co-cultivation, explants were washed and shifted onto SIM containing 2.0 mg l−1 BA and 0.9 mg l−1 mT, which had a higher response to explants responded (82.6%), resulting in 456.3 plants regenerated (Table 4). After that, 148.3 transformed plants were established for hardening (Table 4). Conversely, explants were cultured on SIM with only 2.0 mg l−1 BA, which responded to 62.3% of explants and produced 302 regenerated plants (Table 4). Eventually, 92.6 transformed plants were designated as hardened. After screening for hygromycin B, the Gus assay was performed for all transformed plants. The respective regeneration medium augmented with mT had maximum transformation efficiency (27.3%) (Table 4), while the regeneration system without mT had a lower transformation efficiency (18.6%) (Table 4). Our findings reveal that the combination of mT substantially enhanced transformation efficiency in radish via an optimized regeneration system. Furthermore, there has been no report on the association of mT with improving transformation efficiency in other plants.

Table 4. Influence of mT on A. tumefaciens–mediated genetic transformation efficiency in radish
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GUS assay and molecular confirmation of transformed radish

A distinct blue color was noticed in the transformed explants, including a cotyledonary node (Fig. 4b), shoot induction (Fig. 4d), shoot elongation (Fig. 4f), and rooting (Fig. 4h) that showed the gus A gene was successfully expressed in transformed tissues. The blue color was not found in the NT cotyledonary node (Fig. 4a), shoot induction (Fig. 4c), shoot elongation (Fig. 4e), and rooting explants (Fig. 4g). Previously, the gus A gene was successfully used to establish transformed radish as a reported gene (Cho et al. 2008; Muto et al. 2021). PCR was performed on three gus-positive plants to confirm the presence of the gus A (Fig. 5a, lanes R1–R3) and hpt II (Fig. 5b, lanes R1–R3) genes at about 781 and 1024 bps in the transformed plants and plasmid DNA (Fig. 5a and b, lane P). Nevertheless, amplification was not noticed in the NT plants (Fig. 5a and 5b, lane R4). In RT-PCR, a 1024-bp amplicon was significantly amplified by hpt II gene–specific primers in three PCR-positive plants (Fig. 6a, lanes R1–R3), which confirmed that hpt II is successfully expressed in radish plants. There was no expression in the NT plant (Fig. 6a, lane R4). Furthermore, hpt II expression has been validated by qRT-PCR in three RT-PCR-positive plants (Fig. 6b). In comparison, the NT plant exhibited no expression (Fig. 6b). Our study revealed the maximum level of hpt II expression detected in R3 (Fig. 6b). The lowest level was noticed in R2 (Fig. 6b). Similarly, hpt II is effectively expressed in radish using hypocotyl explants (Muto et al. 2021) as well as a variety of crops such as Arachis hypogaea (Karthik et al. 2018), Kalanchoe laxiflora (Wang et al. 2019), and Momordica charantia (Karthik et al. 2021).

Figure 4.
figure 4

Stable expression of the gus A gene in radish is induced by various regeneration stages of explant. (a) NT cotyledonary node. (b) Transformed cotyledonary node. (c) NT shoot induction. (d) Transformed shoot induction. (e) NT shoot elongation. (f) Transformed shoot elongation. (g) NT plantlet. (h) Transformed plantlet.

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Figure 5.
figure 5

Detection of gus A and hpt II genes integration in transformed radish plants. (a) and (b) PCR analysis. Lane 1—Gene Ruler-1 kb DNA ladder (#SM0313). Lane P—pCAMBIA1301; Lanes R1, R2, and R3—transformed plants. Lane R4—NT plant.

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Figure 6.
figure 6

Expression and quantification level of the hpt II gene in transformed radish plants. (a) RT-PCR analysis. Lane R4—NT plant RNA as a negative control. Lanes R1, R2, and R3—transformed plant RNA samples. (b) qRT-PCR analysis. The relative expression of three transformed lines (R1, R12, and R3) and R4 acting as NT plants was examined by the 2−ΔΔCt method.

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Conclusion

This would probably be the first study to demonstrate the impact of mT on radish regeneration (Fig. 7) and genetic transformation strategies (Fig. 7). Consequently, genetic engineering must establish a solid connection between regeneration and transformation efficiency to raise improved crop varieties for sustainable agriculture. Regeneration efficiency is enhanced by mT incorporated into PGRs. Furthermore, transformation efficiency increased (27.3%) when transformed cotyledonary nodes regenerated on a medium comprising an optimal concentration of 0.9 mg l−1 mT, 2.0 mg l−1 BA, 2.0 mg l−1 GA3, and 0.9 mg l−1 IBA, corresponding to regeneration without mT (18.6%) efficiency. Hence, these strategies could assist in developing essential traits in different radish varieties.

Figure 7.
figure 7

A flowchart demonstrated the regeneration and genetic transformation steps of radish var. Pusa Chetki.

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