Abstract
The present study is an attempt to establish a fast, highly reproducible transformation with a simplifed regeneration system in soybean targeting the apical meristem. The modifed half-seed explants from soybean cultivar (cv.) JS335 were subjected to diferent time intervals of sonication (0, 1, 10, 20, and 30 min) and vacuum infltration (0, 1, 10, 20, and 30 min) in the presence of Agrobacterium tumefaciens strain EHA105 harbouring pCAMBIA1301. The explants were then co-cultivated and subjected to a modifed plant regeneration process that involves only two steps (1) primary shoot regeneration, and
(2) in vitro rooting of primary shoot. The rooted plantlets were hardened and maintained in the greenhouse until maturity. Sonication treatment of 10 min, followed by plant regeneration using a modifed method, recorded the highest transformation efciency of 26.3% compared to other time duration tested. Furthermore, 10 min of vacuum infltration alone resulted in even higher transformation efciency after regeneration, reaching 28.0%. Interestingly, coupling sonication and vacuum
infltration for 10 min respectively produced the highest transformation efciency after regeneration of 38.0%. The putative transformants showed gus expression in mature leaves, trifoliate leaves, fowers, and pods. The presence of hpt II was also confrmed in putative transformants, with an amplicon size of 500 bp. Quantitative real-time PCR confrmed the existence of hpt II as one to two copies in the soybean genome of T0 plants. Furthermore, the segregation pattern was observed in the T1 generation soybean plants which were confrmed using PCR for hpt II. The optimized protocol when tested with other
Indian soybean cultivars showed an enhanced transformation efciency ranging from 19.3% (cv. MAUS47) to 36.5% (cv. CO1). This optimized protocol could provide a reliable platform to overcome the challenges that are associated with the genetic engineering of soybean.
Introduction
Soybean (Glycine max (L.) Merrill), an economically valuable crop, is largely used for consumption and industrial applications (Widholm et al. 2010). The global population growth and the consistent demand for soy products are leading to a continuous increase in the production and demand for soybeans. Consequently, signifcant eforts have been dedicated to improving the regeneration system and the
efectiveness of transforming soybeans. These eforts show great potential for developing superior soybean varieties with desired characteristics. Up until now, soybean regeneration has been achieved through somatic embryogenesis, direct organogenesis, and indirect organogenesis. However, poor regeneration has been a major obstacle in the indirect organogenesis method for soybeans. Most of the research
conducted on soybeans has focused on somatic embryogenesis or direct organogenesis. Regarding soybean transformation, various intrinsic factors such as Agrobacterium strains, types of explants, composition of culture media, duration of co-cultivation, and plant selection markers have been extensively investigated to enhance the efciency of the transformation process. Moreover, extrinsic factors like physical wounding of explants, sonication, and vacuum infiltration have been optimized to achieve higher transformation efciency in soybeans Despite various studies aimed at improving soybean
transformation efciency using Agrobacterium infection, the success rate has been very low due to genotype dependency and low regeneration of transformants (Kumari et al. 2016; Liu et al. 2004). Moreover, poor shoot elongation and long regeneration duration are other important limiting
factors for the efective regeneration of transformants (Ma and Wu 2008). Thus, there is an urgent need to look for alternative ways to develop transformed soybean to meet the global demand. In this regard, the current study aims to develop a fast, reliable, and efcient soybean transformation system incorporating sonication and vacuum infiltration thereby targeting the apical meristem of modifed half-seed
explants. Moreover, the highlight of the present study is the hassle-free and fast regeneration of transformed plants from infected half-seed explants using a simplifed regeneration method that involves just two steps (1) primary shoot regeneration, and (2) in vitro rooting of primary shoot. The optimized protocol has also been tested with 10 cultivars to check its efficiency.
Materials and methods
Indian soybean cultivars (cv.) JS335, PUSA 9712, CO1, TAMS-38, JS71-05, JS93-05, NRC7, MAUS47, PK416,
and Punjab 1 were procured from ICAR-Indian Institute of Soybean Research, Indore, Madhya Pradesh, India, and the cultivars were grown and maintained at the research garden, Department of Biotechnology, Bharathiar University, Coimbatore, Tamil Nadu, India. The optimization was carried out using the soybean cultivar (cv.) JS335 (Fig. 1a). To begin the experiment, the seeds of soybean cv. JS335 were subjected to surface sterilization and imbibed in sterile water for a period of 24 h (Fig. 1b). Following imbibition, the seed coat was removed, and the cotyledons were separated. Only the cotyledon containing the embryonic axis was utilized for the study. Additionally, the radicle of the embryo, which was attached to the cotyledon, was carefully dissected to obtain the modifed half-seed explant (Fig. 1c).
For primary shoot regeneration, explants were inoculated on MS medium supplemented with diferent concentrations of 6-Benzylaminopurine (BAP) (0–8.8 μM) and cultured for 30 days. The explants were sub-cultured into a fresh medium with respective hormonal concentrations at 15 days intervals. For rooting of primary shoots, MS medium supplemented with diferent concentrations of Indole-3 butyric acid (IBA) (0–9.8 μM) was used and the culture was incubated for 30 days. In-order to select the primary shoot after
transformation, minimum inhibitory concentration (MIC) was determined in modifed half-seed explants by inoculating in regeneration medium (MS+2.2 μM BAP; pH 5.7) with diferent concentrations of hygromycin B (0–5 mg l −1) and incubating for 30 days. In addition, the explants were sub-cultured at 15 days intervals. Subsequently, the established primary shoots were transferred to a rooting medium
(MS+4.9 μM IBA; pH 5.7) with diferent concentrations of hygromycin B (0 to 3 mg l −1) and incubated for 30 days for selection at the rooting stage. All the cultures were maintained at 25±2 °C under a 16/8-h photoperiod.
Agrobacterium tumefaciens strain EHA105 harbouring pCAMBIA1301 was used for transformation (Fig. 2). The T-DNA region of the binary vector contains hygromycin phosphotransferase II (hpt II) as the plant selection marker and gus as a reporter gene. The vector backbone carries the neomycin phosphotransferase II (npt II) for bacterial selection. Agrobacterium culture was prepared by inoculating a
single colony into 30 ml LB broth containing antibiotics such as kanamycin (50 mg l −1) and rifampicin (25 mg l −1). The culture was incubated at 28 °C for 16 h at 180 rpm. The bacterial culture was centrifuged at 6000 rpm for 15 min, and the pellet was suspended in a liquid MS medium. Additionally, a flter-sterilized solution of 200 μM acetosyringone was added to the bacterial suspension, which was then
incubated for 1 h at 28 °C at 180 rpm. The absorbance of bacterial suspension was adjusted to 1.0 at OD600 prior to infection. For genetic transformation, the modified half-seed
explants were inoculated into 30 ml Agrobacterium suspension and sonicated for diferent durations (0, 1, 10, 20, and 30 min). Similarly, the explants were subjected to vacuum infltration for diferent time intervals (0, 1, 10, 20, and 30 min) in 30 ml Agrobacterium suspension. Finally, the explants were subjected to combined treatments of sonication (10 min) and vacuum infltration (10 min) in the presence of Agrobacterium. After diferent treatments, the explants were then incubated in fresh Agrobacterium suspension (30 ml) at 28 °C for 30 min. After 30 min, the explants were blot-dried and placed in a co-cultivation medium (MS + 200 μM acetosyringone; pH 5.7) and cultured in complete darkness at 25±2 °C for 3 days. Subsequently, the explants were then thoroughly washed with sterile distilled water containing 350 mg l −1 cefotaxime and cultured in regeneration medium (MS + 2.2 μM BAP + 3 mg l−1 hygromycin B; pH 5.7) for 30 days for selection of primary shoots. The excised primary shoots were then cultured ina rooting medium (MS medium+4.9 μM IBA +2 mg l−1 hygromycin B; pH 5.7) for 30 days. The rooted plantlets were carefully removed from the medium, washed with sterile distilled water, hardened for 2 weeks in paper cups and
Mean values of three independent experiments (±) with standard errors (n=100×3). Values with the diferent letters within columns are signifcantly diferent according to Duncan’s multiple range test (DMRT) at a 5% level
aTotal number of primary shoots survived on regeneration medium (MS+2.2 μM BAP+3 mgl−1 hygromycin B) after 30 days of culture.
bTotal number of primary shoots responded for the root development after 30 days of culture on rooting medium (MS+4.9 μM IBA+2 mg l−1 hygromycin B)
cTotal number of putatively transformed plants that survived in the greenhouse after hardening
dTotal number of putatively transformed plants showing the presence of hpt II
eTransformation efciency=number of hpt II PCR positive plants/total number of infected explants×100
The superscript letter f, g, h, i, j shows that these values are signifcantly diferent according to DMRT
twice to ensure accuracy and reliability. As for the transformation experiments, 100 explants were employed for the respective treatments, and the experiments were repeated three times. The resulting data were presented as mean values with the standard error (SE). Statistical analysis
was performed using SPSS software version 20, specifcally employing Duncan’s multiple range test (DMRT) to determine signifcant diferences at a signifcance level of P<0.05. For segregation ratio analysis, the SE and Chisquare analysis were used (Gomez and Gomez 1984; Hada et al.2018). Signifcance was determined for values with a P<0.05.
Results and discussion
We have conducted studies on various parameters to improve transformation after regeneration efciency in Indian soybean cultivars to address the challenges associated with soybean transformation, including low regeneration rates and the absence of cultivar-independent protocols. In the soybean direct organogenesis system, the explants will be initially subjected to multiple shoot induction. Then
attempts will be made to elongate the shoots, and after elongation, the shoots will be cultured for in vitro rooting. In addition, this process takes approximately 3 or more months to obtain an in vitro rooted plantlet that will be ready for hardening. In order to achieve regeneration using a direct organogenesis system, the radicle, and plumule of the half-seed explants have to be excised and need to be
placed in cytokinin containing medium to trigger the meristematic cells to produce multiple shoots. However, in this method, most of the shoots will fail to elongate affecting the regeneration response (Ether et al. 2013). To overcome this limitation with half-seed explants, we have modified the explant preparation in a way that we removed only the radicle and left the plumule intact to obtain the modifed
half-seed explants. The presence of plumule in the modifed half-seed explants directed the regeneration system towards primary shoot development followed by subsequent in vitro rooting diverting it from the conventional direct organogenesis process that includes multiple shoot induction, shoot elongation, and rooting. Moreover, this diversion also bypassed the shoot elongation step which was critical
in afecting the regeneration efciency. In the present investigation, the primary shoots developed and elongated in the same BAP medium avoiding the necessity of a separate shoot elongation process. Also, using this method, we were able to produce rooted plantlets that are ready for hardening
within 60 days (30 days for primary shoot regeneration and 30 days for rooting) which was comparatively less than similar reports on soybean. The aforesaid advantages of using
Fig. 4 GUS analysis and molecular confrmation of putative transformants regenerated from modifed half-seed explants infected with Agrobacterium tumefaciens strain EHA105 harboring pCAMBIA1301. a Expression of gus in mature leaf from putatively transformed plants; b mature leaf from non-transformed plant; c expression of gus in trifoliate leaves from putatively transformed plants; d trifoliate leaves from non-transformed plant; e expression of gus in fower from putatively transformed plants; f fower from non-transformed plant; g expression of gus in pod from putatively transformed plants; h pod from non-transformed plant; i molecular confirmation for the presence of hpt II in putatively transformed soybean plants. Lane 1: DNA ladder (1 Kb); lane 2: pCAMBIA1301 plasmid (positive control); lane 3: soybean genomic DNA from non-transformed plants (negative control); lanes 4–8: genomic DNA from putatively
transformed soybean plants with expected amplicon (500 bp) of hpt II
this modifed half-seed method also highly favoured efcient regeneration in transformation experiments
In the present study, modifed half-seed explants produced the highest response in inducing the primary shoot regeneration (93.0%) in MS medium supplemented with 2.2 µM BAP (Supplementary Table 1). In addition, the maximum in vitro rooting of primary shoots (75.5%) was observed in the MS medium supplemented with 4.9 µM IBA (Supplementary Table 2). Our fndings were similar to those
of Arun et al. (2015) and Chen et al. (2018), where the aforementioned concentrations of BAP and IBA showed the best response in inducing shoots and roots in soybean. The MIC of hygromycin B in primary shoot regeneration was found to be 3 mg l−1 and the MIC of hygromycin B in in vitro rooting
of primary shoots was 2 mg l−1. The use of hygromycin B as a potent plant selection marker in soybean was established by Olhoft et al. (2006).
In the present study, modifed half-seed explants produced the highest response in inducing the primary shoot regeneration (93.0%) in MS medium supplemented with 2.2 µM BAP (Supplementary Table 1). In addition, the maximum in vitro rooting of primary shoots (75.5%) was observed in the MS medium supplemented with 4.9 µM IBA (Supplementary Table 2). Our fndings were similar to those
of Arun et al. (2015) and Chen et al. (2018), where the aforementioned concentrations of BAP and IBA showed the best response in inducing shoots and roots in soybean. The MIC of hygromycin B in primary shoot regeneration was found to be 3 mg l−1 and the MIC of hygromycin B in in vitro rooting
of primary shoots was 2 mg l−1. The use of hygromycin B as a potent plant selection marker in soybean was established by Olhoft et al. (2006).
In this study, the transformed modifed half-seed explants were successfully regenerated using an optimized regeneration method. Among the diferent treatments applied, the modifed half-seed explants that underwent a 10-min sonication treatment exhibited the highest number of primary shoots that survived (54.6), along with a substantial number of rooted shoots (44.3) and plants that survived
after a 2-week hardening period (26.3). The transformation efciency for this treatment was calculated to be 26.3% (Table 1). However, it is worth noting that the transformation efciency decreased when the sonication time was reduced to 1 min or increased to 20 and 30 min, as indicated in Table 1. The highly active and rapidly dividing meristematic cells that were used for genetic transformation
are present in the primary shoot. Sonication creates microwounds through which Agrobacterium could easily reach the meristematic cells and enhance the transformation efficiency (Trick and Finer 1997). Our study is consistent with the fndings of Hada et al. (2018), where the optimum sonication time was found to be 10 min, and increasing the sonication time decreased the transformation efciency from 36.2% to
12.1%. Guo et al. (2015) also claimed that sonication for 2 s improved transformation efciency in soybean from 2.5 to 5.7%. Vacuum infltration has been well validated as an efcient method to improve the rate of transformation by creating negative atmospheric pressure, enabling easy passage for Agrobacterium to target the meristematic cells (Subramanyam et al. 2013). Among diferent time durations
for vacuum infltration tested (0, 1, 10, 20, and 30 min), a treatment duration of 10 min showed the maximum number of survived primary shoots (51.6), number of rooted shoots (40.6), number of plants that survived after 2 weeks of hardening (28.0), with the transformation efciency of 28.0% (Table 1). Furthermore, it was observed that extending the vacuum infltration time beyond 10 min had a negative impact on the transformation efciency. This decrease in efciency can be attributed to the injury caused to the explants due to excessive vacuum. Similarly, reducing the vacuum infltration time to 1 min resulted in a decreased transformation efciency of 11.6%. In the case of the treatment involving sonication for 10 min combined with vacuum infltration for 10 min, the number of primary shoots that survived in the selection medium was recorded as 54.6 (Fig. 1d and e). Additionally, the number of rooted shoots
was 46.6 (Fig. 1f and g). Following the hardening process, a total of 38.0 plants successfully survived and acclimatized (Fig. 1h and i). Importantly, the transformation efficiency significantly improved to 38.0%, which is considerably higher than the efciency observed in the explants treated with sonication or vacuum infltration alone (Table 1). These fndings align with the previous studies conducted on soybeans by Arun et al. (2015) and Hada et al. (2018), which also suggested that combining sonication and vacuum infltration can enhance the transformation efciency, as demonstrated in the present study.
From the histochemical GUS assay, it was observed that the mature leaf (Fig. 4a), trifoliate leaves (Fig. 4c), flower (Fig. 4e), and pod (Fig. 4g) from putative transformants developed an intense blue colour and tested positive for gus expression. The mature leaf (Fig. 4b), trifoliate leaves
(Fig. 4d), fower (Fig. 4f), and pod (Fig. 4h) from nontransformed plants did not show the gus expression. In the present study, the transformation efciency was calculated based on the presence of the hpt II in transformed plants. The T0 plants that survived after hardening were subjected
to this analysis. The amplicon size of 500 bp (Fig. 4i, Lane 4–8) indicated the presence of hpt II in transformed plants. pCAMBIA1301 plasmid served as the positive control (Fig. 4i, Lane 2) whereas non- transformed plants did not show any amplifcation for hpt II (Fig. 4i, Lane 3). Overall maximum transformation efciency of 38.0% was achieve when modifed half-seed explants were subjected to sonication (10 min) and vacuum infltration (10 min). In this present study, the copy number of hpt II in T0
plants was determined by quantitative real-time PCR using
Mean values of three independent experiments (±) with standard errors (n=100×3). Values with the diferent letters within columns are signifcantly diferent according to Duncan’s multiple range test (DMRT) at a 5% level
aTotal number of primary shoots survived on regeneration medium (MS+2.2 μM BAP+3 mg l −1 hygromycin B) after 30 days of culture
b Total number of primary shoots responded for the root development after 30 days of culture on rooting medium (MS+4.9 μM IBA+2 mg l−1 hygromycin B)
cTotal number of putatively transformed plants that survived in the greenhouse after hardening
dTotal number of putatively transformed plants showing the presence of hpt II
eTransformation efciency=number of hpt II PCR positive plants/total number of infected explants×100
Actin as the reference gene. The results revealed that the copy number of hpt II ranged between one and two. The T0 transgenic soybean lines GmJS335-2 and GmJS335-3 had two copies, while lines GmJS335-1, GmJS335- 4, GmJS335-5, GmJS335-6, GmJS335-7, GmJS335-8, and GmJS335-9 had one copy of hpt II (Supplementary Table 3). The quantitative real-time PCR is replacing the traditional method of detecting the copy number of the foreign gene via the southern blot technique due to various
advantages such as accuracy, lower cost, higher stability, and ease of operation. This technique has been successfully employed in several crops, including cotton (Yang 2012), wheat (Gadaleta et al. 2011), rice (Wei et al. 2011), maize (Yuan et al. 2010), tomato (Wang et al. 2011), and soybean (You-wen et al. 2012). Additionally, the segregation pattern observed in the T1 generation demonstrated Mendelian inheritance with a ratio of 3:1 in one plant (GmJS335 8) at a signifcance level of 0.05% (Supplementary Table 4). The optimized protocol has been further applied to evaluate the transformation efciency in other cultivars of soybean. In the present investigation, cv. JS335 was found to be having the highest transformation efciency of 38.0% followed by the cv. CO1 (36.5%) and cv. TAMS-38 (33.6%). Among the diferent cultivars
examined, cv. MAUS47 displayed the lowest transformation frequency, recorded at 19.3% (Table 2). Overall, the method developed in this study proved to be fast and highly efcient in obtaining transgenic lines within a relatively short duration of 60 days to obtain rooted plantlets. In comparison, previous studies conducted by Arun et al. (2015), Hada et al. (2018), and Wang et al. (2022) demonstrated soybean transformation systems utilizing conventional direct organogenesis, which required longer
regeneration times of 123 days, 104.5 days, and 97 days, respectively. Therefore, this simplifed transformation and modifed regeneration protocol can be utilized effectively for developing transgenic soybean varieties with desired traits.
Conclusion
In this study, we have successfully developed a simple regeneration system from modifed half-seed explants, consisting of two steps: primary shoot regeneration and rooting. This system has been efectively adapted to regenerate transgenic plants from modifed half-seed explants infected with Agrobacterium, and it ofers the advantage of a shorter regeneration period. Additionally, the incorporation of sonication and vacuum infltration techniques has signifcantly enhanced the transformation efciency in our study. Moreover, this transformation and regeneration system has demonstrated its efcacy across various soybean cultivars, indicating its wide applicability. We believe that this simple protocol holds great potential for commercial trait improvement in diverse soybean varieties.
Supplementary Information The online version contains supplementary material available at https://doi.org/10.1007/s13205-023-03715-8
Acknowledgements The authors are thankful to the University Grants Commission: Basic Startup Research Grant (No.F.30-410/2018 (BSR); dt.: 29.06.2018), New Delhi, Government of India, for fnancial assistance provided to Muthukrishnan Arun, and also thanks to ICAR -Indian Institute of soybean research, Indore, Madhya Pradesh, India for providing soybean seeds to carry out the research work.
Author contributions MA and KS: Conception and idea. KS: performed the experimental work with the assistance of MA. NV and KS wrote the manuscript with the assistance of MA. KS and NV: preparation of tables and fgures. CA: helped in performing experimental analysis. PG and CA: helped in the critical reviewing of the manuscript. Finally, all the authors approved for the manuscript.
Data availability Data generated in this work is included in the manuscript and in the supplementary material. This will be made available on request.
Declaration
Conflict of interest There are no conficts of interest to declare.
Ethical approval for involving human participants and/or animals Not applicable, since this article does not contain any studies with human participants or animals performed by any of the authors.
Author Name
Dr. Krishnagowdu Saravanan