An optimized protocol for in vitro regeneration and genetic transformation of broomcorn millet

doi:10.21203/rs.3.rs-4697063/v1

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Research Article

An optimized protocol for in vitro regeneration and genetic transformation of broomcorn millet

https://doi.org/10.21203/rs.3.rs-4697063/v1

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Background

Broomcorn millet has many advantages, such as abiotic stress resistance, a short growth cycle and high nutritional value. However, due to the lack of efficient genetic transformation methods for broomcorn millet, the characterization of genes related to important traits lags behind that of other crop species. Therefore, establishing efficient in vitro regeneration and genetic transformation methods for broomcorn millet is essential.

Results

In this study, we used mature seeds from the sequenced cultivar 'Longmi 4' as explants and optimized their in vitro regeneration and genetic transformation methods. The optimal hormone concentrations for embryogenic callus induction medium were 2.5 mg/L 2,4-D and 0.5 mg/L BAP. The optimal hormone concentrations for shoot regeneration media were 2 mg/L kinetin and 0.5 mg/L a-naphthaleneacetic acid. Additionally, the cocultivation time was 3 days, and the optimal hygromcin concentration for putative transgenic callus selection was 45 mg/L. The transgenic efficiency was 21.25% after our modification approach.

Conclusions

Here, we present a simple and highly efficient Agrobacterium-mediated genetic transformation protocol for broomcorn millet. Our work provides a tool for the characterization of genes related to important traits, as well as a new strategy for broomcorn millet breeding.

Broomcorn millet (Panicum miliaceum L.), or proso millet, which belongs to the Poaceae family, originated from northern China approximately 10 000 years ago [1]. It is cultivated in arid and semiarid areas with a high degree of drought and salt tolerance [2–4]. It also has a short growth cycle, with early-maturing varieties reaching maturity in 60 days [5]. In addition, its grains have high nutritional and medicinal value [6–8]. However, due to the lack of efficient genetic transformation methods for broomcorn millet, the characterization of genes related to these important traits lags behind that of other crop species.

Agrobacterium-mediated genetic transformation is a natural plant genetic transformation system characterized by low copy numbers and genetic stability of exogenous genes in plants [9]. Initially, used for transgenic dicot plants, significant progress has been made over the past two decades in major graminaceous crops such as rice, maize, wheat, and other grasses [10]. Rice, as a model monocot plant, has undergone relatively systematic genetic transformation since the first Agrobacterium-mediated transformation in 1993 [11]. Researchers have optimized this system, achieving transformation efficiencies of up to 90% [12, 13]. Unlike rice, genetic transformation in maize is greatly influenced by the type of callus and genotype, with immature embryos and the maize cultivar A188 being more easily transformed. Adjusting the spatial and temporal expression patterns of the Baby boom (Bbm) and Wuschel2 (Wus2) genes has increased transformation efficiency in several commercial maize varieties, independent of genotype [14]. Other graminaceous crop species, including wheat and sorghum, still exhibit relatively low genetic transformation efficiencies. Kumar et al. [15] generated callus tissue from immature and mature bread wheat embryos, infected them with Agrobacterium, and successfully cultivated stable transgenic plants, with transformation efficiencies of 14.9% in immature embryos and 9.8% in mature embryos. Similarly, the genetic transformation efficiency of sorghum is strongly influenced by its genotype, with transformation rates ranging from 0.3–8.3% [16].

In vitro regeneration and genetic transformation of broomcorn millet are still in the early stages. Only a few research teams have attempted to construct in vitro regeneration systems using different explants. In 1973, Rangan et al. [17] first used the mesocotyl as an explant to culture broomcorn millet in vitro, successfully inducing embryogenic callus but failing to obtain regenerated plants. Later, Rangan and Vasil [18] induced embryogenic calli using floral primordia from young panicles and successfully obtained complete plants. Recently, Liu used mature embryos from broomcorn millet seeds as explants, transformed them with Agrobacterium, and successfully established a genetic transformation system [19, 20]. Liu reported significant differences in regeneration ability among different plant genotypes. The average transformation efficiency for 'Longmi 4', which is a widely cultivated genotype and a sequencing cultivar, was 4%. The low efficiency greatly limits the application of advanced technologies such as gene editing in this crop. Therefore, establishing efficient in vitro regeneration and genetic transformation methods for broomcorn millet is essential.

In this study, we used 'Longmi 4' mature seeds as explants, compared the components of the tissue culture media, optimized the in vitro regeneration system of broomcorn millet, and explored two important factors affecting Agrobacterium-mediated transformation. The goal was to establish a highly efficient in vitro regeneration and genetic transformation system for broomcorn millet.

Plant Materials

Mature seeds of the broomcorn millet cultivar 'Longmi 4' used in this study were obtained from the College of Agriculture, Inner Mongolia Agricultural University.

Embryogenic callus induction

The dry seeds were carefully dehusked with abrasive paper. Dehusked and healthy seeds were sterilized in 75% (v/v) ethanol for 1 min, followed by 10% sodium hypochlorite for 5 min, after which they were washed with sterilized water five times and subsequently air-dried on sterile filter paper. The sterilized seeds were then inoculated on callus induction medium (MCI) adapted from rice [21]. The MCI medium consisted of MS salt containing all vitamins, 300 mg/L casein enzymatic hydrolysate, 600 mg/L L-proline, 30 g/L maltose, and 3 g/L Phytagel. The pH was adjusted to 5.8 with 0.1 M NaOH before autoclaving. To identify the optimal callus induction medium for 'Longmi 4', four different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyla minopurine (BAP) were tested using an orthogonal array L16(4²) (Table 1). Twenty seeds were placed on each Petri dish, and 5 plates were used for each treatment. The plates were incubated at 26°C ± 2°C in the dark. Primary calli appeared after 2 weeks, and embryogenic calli were obtained after subculturing for another 2 weeks. The embryogenic callus induction rate was calculated as (number of embryogenic calli/number of seeds) × 100%.

Table 1

Orthogonal experiment results of embryogenic callus induction
Treatments	Factors		Induction rate(%)
Treatments	2,4-D (mg/L)	BAP (mg/L)	Induction rate(%)
1	1(2.0)	1(0.0)	30.00%
2	1	2(0.25)	33.35%
3	1	3(0.5)	46.65%
4	1	4(1.0)	40.30%
5	2(2.5)	1	46.65%
6	2	2	61.25%
7	2	3	66.66%
8	2	4	63.35%
9	3(3.0)	1	50.00%
10	3	2	46.70%
11	3	3	56.65%
12	3	4	46.70%
13	4(3.5)	1	36.65%
14	4	2	40.00%
15	4	3	56.65%
16	4	4	23.35%

Plant regeneration

The embryogenic callus was then transferred to shoot regeneration medium (SRM) to induce shoot formation. The SRM consisted of MS salt with vitamins, 2 mg/L kinetin, 0.5 mg/L a-naphthaleneacetic acid (NAA), 30 g/L maltose, and 3 g/L Phytagel, pH 5.8. To optimize the cytokinin concentration in SRM media, kinetin and NAA were tested at four different concentrations using an L16(4²) orthogonal array (Table 2). Ten embryogenic calluses were placed on each Petri dish, and 3 plates were used for each treatment. The plates were incubated at 26°C ± 2°C under a 16/8 h light/dark cycle. After 2 weeks, the shoot grew to 1–2 cm, and the shoot was transferred to rooting media consisting of half-strength MS salt supplemented with 30 g/L sucrose and 3 g/L Phytagel. The regeneration rate was calculated as (number of regenerated plants/number of embryogenic calli) × 100%.

Table 2

Variance (ANOVA) analysis of the results of the orthogonal experiment results
Hormones	SS	DF	MS	F	P
2,4-D	0.126	3	0.042	8.498	0.005**
6BA	0.058	3	0.019	3.951	0.047*
Residual	0.044	9	0.005

Binary vector

The binary vector pRHVcGFP was kindly provided by Dr. Wang Guoliang [22]. Green fluorescent protein (GFP) was used as the reporter gene under the control of the maize ubiquitin (Zmubi) promoter, and the hygromycin phosphotransferase (hpt) gene was used as the selection marker gene under the control of the cauliflower mosaic virus 35S promoter.

Determination of hygromycin concentration

To determine the appropriate hygromycin concentration for selecting putative transformants, the calli were cultured in MCI media supplemented with different hygromycin concentrations (0, 10, 15, 20, 25, 30, 35, 40, 45, and 50 mg/L). Ten calli were cultured on a Petri dish plate (90 mm), and each treatment was repeated three times. After 4 weeks of cultivation at 26°C ± 2°C in the dark, the survival rate of the calli was recorded.

Genetic transformation

The transformation method used in this study was modified from a rice transformation protocol [21]. pRHVcGFP was transformed into Agrobacterium tumefaciens EHA105. Single colonies were scraped from the plate and inoculated into 100 mL of LB liquid media supplemented with 50 mg/L kanamycin and 35 mg/L rifampicin at 28°C and 200 rpm until an OD of 1.0 was reached. The cells were then centrifuged at 5000 × g for 10 min and resuspended in 100 ml of inflation medium (MS salt, 68 g/L sucrose, 36 g/L glucose, 3 g/L KCl, 4 g/L MgCl₂, pH 5.2) supplemented with 150 µM acetosyringone. The final OD value was adjusted to 0.5. The suspension culture was incubated at 28°C and 200 rpm for 1 hour.

The embryogenic calli were immersed in suspension medium for 30 min and then dried on sterile filter paper. The infected calli were placed in cocultivation media (RSM media supplemented with 150 µM acetosyringone, pH 5.2) and incubated in the dark at 27°C for 1, 3, or 5 days. After the cocultivation period, the calli were washed 3 times with 300 mg/L of Timentine in sterile distilled water, dried on sterile filter paper, and then transferred to RSM media supplemented with 50 mg/L hygromycin and 300 mg/L Timentine in the dark at 27°C. After 3–4 weeks of selection, the healthy and proliferated calli were transferred onto shoot regeneration media supplemented with 20 mg/L hygromycin or 300 mg/L Timentine and cultured at 26°C under a 16/8 h light/dark cycle for approximately 4 weeks. Subsequently, adventitious shoots were transferred to rooting media supplemented with 300 mg/L of Timentine (pH 5.2) for root induction. After another 4 weeks, the plantlets with well-developed roots were transplanted into soil to generate seeds at 26°C under a light/dark cycle of 12 h/12 h.

PCR analysis and GFP observation of transgenic plants

Genomic DNA from leaves was extracted according to the protocol of the TAINGEN Plant Genomic DNA Miniprep Kit (DP305, Tiangen, Beijing). PCR amplification was performed using the vector-specific primers 35S-hpt-F/35S-hpt-R (35S-hpt-F: CGGTCGGCATCTACTCTATT and 35S-hpt-R: AAACCTCCTCGGATTCCATT) to confirm that the transgenic seedlings were positive. The PCR mixture consisted of 50–100 ng of DNA template, 2.5 µl of 2× buffer, 1 µl of each primer and 0.5 µl of Taq DNA polymerase unit. The volume was adjusted to 25 µl with sterile double distilled water. The PCR products were subjected to gel electrophoresis on a 1% (w/v) agarose gel. For GFP observation, the stems and roots of the transgenic plants were viewed and photographed under blue light (450 nm) with a blue light source (LUYOR-3415RG).

Statistical analysis

All the data are presented as the means ± SDs from three repeated experiments. The transformation rate was calculated as (number of positive transgenic seedlings/number of embryogenic calli) × 100%.

Optimization of embryogenic callus induction medium

To identify the optimal embryogenic callus induction medium for 'Longmi 4', 2,4-D and BAP were tested at four different concentrations using an orthogonal array L16(4²) (Table 1). Different hormone concentrations and combinations significantly affected the CIR. Treatment 7 achieved the highest embryogenic callus induction rate of 66.66%, with concentrations of 2.5 mg/L 2,4-D and 0.5 mg/L BAP. The lowest induction rate of 23.35% was observed at concentrations of 3.5 mg/L 2,4-D and 1.0 mg/L BAP. Interestingly, 2.0 mg/L 2,4-D without BAP produced only a 30% induction rate. Two-way ANOVA was carried out, and the results showed that 2,4-D had a highly significant impact on the CIR, while BAP had a significant effect (Table 2). The optimal CIM was determined to be 4.33 g L^− 1 MS salt, 300 mg L^− 1 casein enzymatic hydrolysate, 600 mg L^− 1 L-proline, 30 g L^− 1 maltose, 3 g L^− 1 Phytagel, 2.5 mg L^− 1 2,4-D, and 0.5 mg L^− 1 BAP at pH 5.8.

Optimization of shoot regeneration medium

To optimize the cytokinin concentration in SRM media, kinetin and NAA were tested at four different concentrations using an L16(4²) orthogonal array (Table 3). Ten embryogenic calluses were placed on each Petri dish (90 mm), and 3 plates were used for each treatment. The regeneration rate of shoot regeneration varied significantly with different treatments (Table 3). The highest regeneration rate from treatment 5 (2.5 mg L^− 1 2,4-D and 0.5 mg L^− 1 BAP) reached 59.55%, while the lowest was 18% in treatment 5. The statistical analysis showed that 2,4-D had a significant effect on the shoot regeneration rate, while NAA was not effective (Table 4). The optimal shoot regeneration media were thus determined to be 4.33 g/L MS, 30 g/L sucrose, 3 g/L Phytagel, 2.0 mg/L kinetin, 0.5 mg/L NAA, and 15% coconut milk at pH 5.8.

Table 3

Orthogonal experiment results of shoot regeneration
Treatments	Factors		Induction rate(%)
Treatments	NAA(mg/L)	Kinetin(mg/L)	Induction rate(%)
1	1(0.0)	1(1.0)	18.00
2	1	2(1.5)	25.00
3	1	3(2.0)	38.46
4	1	4(2.5)	33.33
5	2(0.25)	1	20.00
6	2	2	23.81
7	2	3	45.45
8	2	4	31.25
9	3(0.5)	1	41.67
10	3	2	33.33
11	3	3	59.55
12	3	4	45.45
13	4(1.0)	1	27.78
14	4	2	29.41
15	4	3	41.25
16	4	4	26.31

Table 4

Variance (ANOVA) analysis of the results of the orthogonal experiment
Hormones	SS	DF	MS	F	P
NAA	757.179	3	252.393	6.733	0.011
BAP	957.559	3	319.186	8.515	0.005
Residual	337.379	9	37.487

Determination of hygromycin concentration for selection

To determine the optimal hygromycin concentration for selecting putative transformants, the callus pieces were cultured on MCI media supplemented with different concentrations of hygromycin (0, 10, 15, 20, 25, 30, 35, 40, 45, and 50 mg/L) (Fig. 1). After culturing for 4 weeks, the calli on media supplemented with 45 mg/L or 50 mg/L hygromycin were browned to death, while the calli on media supplemented with no hygromycin continued to proliferate (Fig. 1A, I and J). From 10 mg/L to 40 mg/L, the calli became brown, but some of the calli survived (Fig. 1B-H). The survival rate decreased as the concentration increased (Fig. 2). Therefore, our results indicate that 45 and 50 mg/L hygromycin are suitable for selecting putative transformants.

Determination of cocultivation time

Previous studies have indicated that increasing the cocultivation time enhances Agrobacterium transformation efficiency but results in Agrobacterium outgrowth [23–25]. To determine the optimal balance between transformation efficiency and contamination, cocultivation periods of 1, 3, and 5 days were tested. The number of calli that generated hygromycin-resistant shoots was recorded, and the results showed that a cocultivation period of 3 days produced the greatest number of transgenic shoots (Fig. 3A and 3B). Compared with the two conditions, 5 days of cocultivation induced more contamination (Fig. 3C). A greater transformation rate was obtained after 3 days (Fig. 4).

Genetic transformation

Mature 'Longmi 4' seeds were used as explants for callus induction (Fig. 5A). White and friable embryogenic calli were obtained after culture on MCI media for 4–6 weeks (Fig. 5B). embryogenic calli were immersed in liquid suspension media for 30 min and then incubated in cocultivation media at 22°C for 3 days in the dark (Fig. 5C and 5D). The infected calli were transferred to selection media supplemented with 50 mg/L hygromycin. After 3–4 weeks of selection, the successfully transformed callus appeared white and healthy, while the nontransformed tissue had browned until death (Fig. 5E). Hygromycin-resistant calli were transferred to shoot regeneration media supplemented with 10 mg/L hygromycin at 26°C under a 16 h/8 h light/night cycle for 4–5 weeks (Fig. 5F). Subsequently, adventitious shoots were transferred to rooting media for root induction (Fig. 5G). After 2–3 weeks, the plantlets with well-developed roots were transplanted into the soil, and the seeds were harvested after 6 to 8 weeks (Fig. 5H). The complete transformation cycle lasted approximately 18 to 25 weeks.

Screening and GFP observation of transgenic plants

PCR amplification of a 1424 bp fragment was performed to characterize 12 putative transgenic plants. The pRHVcGFP vector plasmid and wild-type genomic DNA were used as positive and negative controls, respectively. As shown in Fig. 6, 11 lines were confirmed to be positive transgenic plants. In addition, GFP fluorescence was detected in the roots and stems of these transgenic plants (Fig. 7). Green fluorescence could not be detected in the leaves of the transgenic plants because of chlorophyl1 accumulation. We performed three rounds of transformation, and the average transformation rate was 21.25% (Table 5).

Table 5

Orthogonal experiment results of embryogenic callus induction
Experiment	1	2	3
Number of infected calli	80	80	80
Number of regeneration calli	38	40	39
Positive plants	13	17	15
Transformation efficiency (%)	16.25	21.25	18.75
Regeneration efficiency(%)	47.5	50	48.75

Plant intro regeneration is sometimes highly genotype specific. Different regeneration rates were found among different broomcorn millet genotypes [19]. Some cultivars, such as 'Hongmi' and 'Qingyanghongying', had much greater embryogenic callus induction rates than 'Longmi 4', the elite germplasm and a sequencing cultivar. Therefore, in this study, we used mature 'Longmi 4' seeds as explants and optimized their in vitro regeneration and genetic transformation methods.

Optimization of the in vitro regeneration system

Several research teams have attempted to construct in vitro regeneration systems for brromcorn millet using different explants, such as mesocotyl, immature inflorescence and mature seeds [16–19]. Mature seeds with high regeneration efficiency have been used for in vitro regeneration of several monocots, such as rice, sorghum and foxtail millet [16, 21, 23].

In this study, we used mature seeds as explants due to their convenience and flexibility. The type and concentration of phytohormones are critical factors affecting the efficiency of embryogenic callus induction in in vitro plant regeneration [23–25]. In rice and foxtail millet, a combination of auxin (2,4-D) and cytokinins (BAP or NAA) was more effective than auxin (2,4-D) alone. In this study, 2,4-D and BAP were tested at four different concentrations using an orthogonal array L16(4²) (Table 1). Statistical analysis of this orthogonal experiment showed that 2,4-D had a highly significant impact on the CIR (Table 2). We found that a higher concentration of 2,4-D generated a greater induction rate.

Shoot regeneration efficiency is a major bottleneck in broomcorn millet. In a recent study of broomcorn millet genetic transformation, the regeneration efficiency was 3.5%-10%, and 3.0 mg/L BAP and 0.2 mg/L 2,4-D were used in the regeneration media[20]. Kinetin and NAA were used for the induction of rice shoot regeneration, resulting in a 54–77% regeneration frequency [12]. In our study, the regeneration rate increased to 59.55% when 2.0 mg/L kinetin and 0.5 mg/L NAA were used for shoot induction.

Optimization of Agrobacterium-mediated genetic transformation in broomcorn millet

Agrobacterium-mediated transformation is widely used in plant genetic transformation due to its high efficiency, effectiveness, simplicity, genetic stability, and low cost [9]. It is extensively applied in several monocot species, such as rice, maize, sorghum, and foxtail millet [12–16, 23]. However, genetic information on the transformation of broomcorn millet is still limited. The Agrobacterium-mediated genetic transformation methods should be optimized for broomcorn millet, especially for the sequenced cultivar 'Longmi 4'.

Various studies have attempted to increase the efficiency of genetic transformation in major crop species and could be applied in broomcorn millet [9–12]. In this study, we optimized the cocultivation time and hygromycin concentration for selection. The optimal duration of cocultivation is crucial for facilitating T-DNA transfer with the assistance of acetosyringone [24]. In this study, 3 days of cocultivation was determined to be the optimal cocultivation period, which was consistent with the results in other graminaceous plants [25–28].

The selection of an appropriate screening agent concentration is crucial for successful Agrobacterium-mediated transformation of embryogenic calli. During the transformation process, only a few embryogenic callus tissues can accept and integrate exogenous DNA, making the selection of transgenic callus tissue vital for effectively detecting transgenic components in regenerated plants. Additionally, antibiotic selection is critical for the success rate of transgenic plants. Excessively high concentrations of hygromycin can cause browning and death of embryogenic callus tissues, whereas concentrations that are too low may not achieve effective selection. Different species require varying selection conditions; for example, foxtail millet uses lower hygromycin concentrations (8 mg/L)[27], while switchgrass requires higher concentrations (75 mg/L)[28]. We tested various hygromycin concentrations ranging from 10 to 50 mg/L and ultimately selected 45 mg/L based on comparative results. We performed three rounds of transformation, and the average transformation rate was 21.25%.

In this study, we provide an optimized protocol for in vitro regeneration and genetic transformation of broomcorn millet. Mature 'Longmi 4' seeds were used as explants. We established an efficient in vitro regeneration method by optimizing the type and concentration of phytohormones in the media and obtained a callus induction rate of 66.5% and a shoot regeneration rate of 56.7%. Additionally, the cocultivation time was 3 days, and the optimal hygromcin concentration for putative transgenic callus selection was 45 mg/L. The transgenic efficiency was 21.25%, which is much greater than that reported in previous studies. Our work provides an efficient tool for the characterization of genes related to important traits, as well as a new strategy for broomcorn millet breeding.

2,4-D

2,4-dichlorophenoxyacetic acid

BAP

6-benzylaminopurine

NAA

a-naphthaleneacetic acid

Murashige and Skoog Medium

MCI

callus induction medium

SRM

shoot regeneration medium

GFP

green fluorescent protein, hpt:hygromycin phosphotransferase

Bbm

Baby boom

Wus2

Wuschel2.

Acknowledgements

The authors would like to thank Dr. Shihua Guo, who provided 'Longmi 4' seeds for our experiment. Dr. Guoliang Wang for kindly sharing the binary vector pRHVcGFP.

Funding

This study was supported by Natural Science Foundation of Inner Mongolia (2021ZD04), The Central Government Guiding Special Funds for the Development of Local Science and Technology (2020ZY0005), Key Research and Development Program of Shanxi Province (2022ZDYF110), Special Project on Crop Germplasm Resources Protection and Utilization of Ministry of Agriculture (19240451), and National Science and Technology Resource Sharing Service Platform Project (NCGRC-2024-27).

Availability of data and material

All data generated or analysed during this study are included in this manuscript.

Authors’ contributions

ZLC, JQJ, DML, and LZ contributed to the writing of the paper. ZLC, WMW, XQH, and YCW developed and carried out the experiments. HGW, LLL, LW, and JL assisted in experimental design and data analysis. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Not applicable.

Ethics approval and consent to participate

Not applicable.

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No competing interests reported.

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An optimized protocol for in vitro regeneration and genetic transformation of broomcorn millet

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Abstract

Background

Results

Conclusions

Figures

Introduction

Materials and Methods

Plant Materials

Embryogenic callus induction

Plant regeneration

Binary vector

Determination of hygromycin concentration

Genetic transformation

PCR analysis and GFP observation of transgenic plants

Statistical analysis

Results

Optimization of embryogenic callus induction medium

Optimization of shoot regeneration medium

Determination of hygromycin concentration for selection

Determination of cocultivation time

Genetic transformation

Screening and GFP observation of transgenic plants

Discussion

Optimization of the in vitro regeneration system

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1