1. Introduction
Erigeron breviscapus (Vant.) Hand-Mazz is a perennial herbaceous plant in the Asteraceae family. This plant is distributed in Hunan, Guangxi, Guizhou, Sichuan, Yunnan, and Tibet provinces in China. It can only be seen in some open slope grasslands and forest margins at altitudes of 1200–3500 m [
1] and has been listed as a nationally protected species of traditional Chinese medicine. The chemical components in
E. breviscapus mainly include flavonoids, caffeoyls, coumarins, lignans, terpenoids, and other components [
2], among which scutellarin has the highest content. Studies have shown that scutellarin can reduce neuronal damage caused by traumatic brain injury, cerebral ischemia, reperfusion injury and hypoxic-ischemic brain injury [
3,
4,
5], and is commonly used to treat acute cerebral infarction in elderly patients [
6] and is also effective in patients with diabetic nephropathy [
7,
8]. Another active ingredient is caffeoylquinic acids (CQAs), which can dilate blood vessels and inhibit thrombosis in vivo [
9].
E. breviscapus is currently the best natural biological medicine for the treatment of obliterative cerebrovascular diseases and the sequela of cerebral hemorrhage, with an efficiency of >95% and slight side effects. According to the World Health Organization, the number of deaths from cardiovascular diseases will increase to 23.6 million by 2030 [
10]. Therefore,
E. breviscapus is of great significance for recovery and secondary prevention after the first onset of cardiovascular and cerebrovascular diseases. In addition,
E. breviscapus is a natural neuroprotective agent for Alzheimer’s disease [
1]. At present, according to different doses and routes of administration of
E. breviscapus extract, various dosage forms have been developed, such as tablets, capsules, oral liquids, and injections, and international research on
E. breviscapus unilateral preparations has also begun. It is expected to become an internationally recognized botanical medicine after ginkgo biloba preparations.
At present, scutellarin is mainly derived from extracts of
E. breviscapus, but the cost of planting
E. breviscapus remains high due to the limited cultivation area, low content of active ingredients, serious pests and diseases, and the degradation of germplasm variation. A yeast cell factory for the total synthesis of scutellarin has been successfully constructed in the
Saccharomyces cerevisiae chassis cells, and the total synthesis of scutellarin has been achieved, but it is still a certain distance from industrial production [
11]. Therefore, urgent measures are needed to develop varieties with high content of active ingredients, high yield, and disease resistance to achieve sustainable utilization of resources.
The development of genetic manipulation, DNA technology, and genetic transformation provides a reliable route for the development of transgenic medicinal plants that are tolerant to abiotic stress, resistant to pests, and have excellent agronomic traits [
12]. Direct genetic transformation has become the method of choice for basic plant research and the primary technique for generating transgenic plants [
13,
14]. Therefore, the development of genetic transformation technology provides an opportunity to accelerate the various improvements of
E. breviscapus.
The two main methods of plant genetic transformation are
Agrobacterium-mediated transformation and biolistic-mediated transformation [
15,
16]. Particle bombardment is simple to perform and is virtually unlimited in terms of plant ranges and material genotypes [
17]. Various types of transformation receptors are suitable for large-sized genetic cargo [
18]. Target genes can be introduced into the organelles of plant cells. Therefore, the biolistic-mediated transformation is widely used in the current transgenic research.
Agrobacterium-mediated transformation is more efficient in dicots than in monocots and is limited to specific plant host ranges [
15,
19,
20]. On the other hand,
Agrobacterium-mediated transformation is generally considered to be more precise and controllable than particle bombardment; however, a report on bombardment strategies [
13,
21] showed little evidence of major differences in levels of transgene instability and silencing when compared in the same species as in other non-model systems. Both approaches have their advantages and limitations [
22,
23]. At present, regenerated plants of different explants of
E. breviscapus, i.e., true leaves [
24], petioles [
25], and anther [
26], have been successfully obtained and plant regeneration programs have been established. He et al. established an
Agrobacterium-mediated transformation system and applied it to the functional verification of biosynthetic pathway genes [
27]. Qiu screened suitable acceptor materials for gene gun induction [
28].
In this study, the leaves of E. breviscapus were used as explants, and different particle bombardment parameters were optimized using an orthogonal design. After range analysis and variance analysis, the main influencing factors were identified, and the experimental effects were accurately and comprehensively evaluated to obtain the optimal transformation protocol. This is a successful attempt to directly transform E. breviscapus by gene transfer using the particle gun, which fully demonstrates the possibility of stable transformation of E. breviscapus. This study has long-term significance for the genetic engineering research of medicinal plants.
3. Discussion
One of the most effective methods for direct gene transfer is the particle bombardment method. Bacteria are not required during the transformation process. It is widely used and efficient in DNA transfer in mammalian cells, microbes, and monocots [
29]. So far, particle bombardment has been used in many medicinal plants, i.e.,
Catharanthus roseus [
30],
Hypericum perforatum [
31],
Centella asiatica [
32],
Tripterygium wilfordii [
33],
Momordica charantia [
34],
Scoparia dulcis [
35], etc. In this study, a simple and effective biolistic transformation method was established in
E. breviscapus with a transformation efficiency of 36.7%.
The use of orthogonal design can reduce the number of experiments and the complexity of experimental analysis methods, overcome the blindness in condition optimization, and improve work efficiency and experimental accuracy. The method of biolistic transformation of
E. breviscapus was optimized by the hybrid orthogonal design. The main influencing factors were precipitation agents, target tissue distance, and plasmid DNA concentration. The optimum transformation conditions were precipitant agents MgCl
2 + PEG, target tissue distance 9 cm, helium pressure 650 psi, bombardment once, plasmid DNA concentration 1.0 μg·μL
−1, and chamber vacuum pressure 27 mmHg. At present, a universal optimization strategy (
Figure 1) has been developed and applied to a variety of medicinal plants, including
Erigeron breviscapus,
Salvia miltiorrhiza,
Tripterygium wilfordii [
33], and
Aconitum carmichaelii. The explants used include leaves, stems, suspension cells, and calluses.
By optimizing the pre-incubation time, we found that 7–14 days of pre-cultivation was more conducive to the regeneration of explants after bombardment. However, shorter (2–5 d) or longer (21 d) pre-culture times were unfavorable to explants’ regeneration. The same conclusion was reached in the biolistic transformation of wheat-microspore-derived calluses and microspores. Pre-culture for 3–8 days could improve the GUS expression in microspores [
36]. The results of optimization experiments of
Fistulifera solaris showed that the highest conversion rate was achieved by pre-culturing for 48 h [
37]. The above results indicated that the optimal pre-cultivation time varied with individual differences. Therefore, it is necessary to optimize the pre-culture time.
The orientation of the plant tissue explants placed on the medium is another important factor affecting the efficiency of plant transformation and regeneration [
38]. The abaxial orientation of the leaf implies that the lower surface of the leaf is in contact with the medium, while the adaxial position means that the upper surface of the leaf is in contact with the medium [
39]. In our pretest study, we found that more adventitious buds were produced during the regeneration stage when the abaxial side of the leaf was exposed to the medium than the adaxial side. Mazumdar et al. reported that the regeneration efficiency of the abaxial end of explants exposed to the medium was twice as high as that of the adaxial end [
40]. Similarly, tomatoes of two different genotypes exhibited higher regeneration efficiency and higher shoot numbers per explant when placed abaxially than when exposed to medium in the adaxial direction [
41], as confirmed in earlier studies [
42,
43].
The PEG/Mg
2+ coating protocol in this study showed better stabilization of the transformation than the standard Spd/Ca
2+ method. Due to the hygroscopicity, oxidizable nature, and deamination over time of spermidine (Spd) solutions, frozen aliquots should be replaced for fresh at least monthly [
44]. Stock solutions of CaCl
2 and Spd must be used separately, whereas PEG and MgCl
2 can be prepared conveniently as a single stock solution that is stable for many years when stored at −20 °C. The PEG/Mg
2+ procedure has been successfully applied to wheat for stable transformation [
45].
The distance from the microcarrier to the target tissues can affect the velocity of the microparticles and thus the frequency of transformation [
46]. As in previous studies, a target tissue distance of 9 cm was reported as the optimal propagation distance for bananas [
47], wheat [
48], and cumin [
49]. We found that a target tissue distance of 9 cm was a significant factor in improving transformation frequency. This distance can reduce damage to a great extent and ensure the distribution of DNA microcarriers on target tissues [
50].
Similar to Jähne et al., we found that changing the helium pressure in the range of 1100–1350 psi had no significant effect on the number of positive shoots [
51]. In contrast to the results of this study, Jähne et al. found that lower (450–900 psi) or higher (2000–2200 psi) pressures reduced the frequency of transformation. When Harwood et al. used barley microspores pre-incubated for 1–4 days, they found that a lower pressure of 450 psi increased the number of GUS-positive microspores [
52]. We also found that lower bombardment pressures increased the number of positive shoots. When Mentewab et al. compared the effect of using 650 psi or 1100 psi pressure, transient expression of microspores in culture for 1 day was observed only after using low pressure, while multicellular structures of 8 days were only observed when high pressure was applied [
53]. It was demonstrated that the optimal helium burst pressure may depend on several factors related to the cell wall properties and the damaging effect of the treatment.
Comparing the number of surviving shoots, an overall optimal parameter was observed, i.e., precipitant agents MgCl2 + PEG, target tissue distance 9 cm, helium pressure 650 psi, bombardment once, plasmid DNA concentration 1.0 μg·μL−1, and chamber vacuum pressure 27 mmHg. In this study, the frequency of transformation obtained by biolistic transformation was 36.7%. The transformation efficiency of E. breviscapus was not reported for either Agrobacterium-mediated or particle bombardment transformation (three adventitious shoots were obtained). The higher transformation efficiency of the particle gun bombardment may be due to the attempted use of the hybrid orthogonal methods. Optimization strategies are highly efficient compared to the previous single-variable method, thus potentially facilitating genetic modification for improved traits.
4. Materials and Methods
4.1. Plant Material and Culture Conditions
Seeds of
E. breviscapus were manually rubbed to remove their pappus, and the plump and undamaged seeds were selected for the experiment. Before inoculation, seeds were soaked for 4 h, dried for 3 h, and then sterilized. Under sterile conditions, seeds were soaked in 75% (
v/
v) ethanol for 8~10 s and washed 3~4 times with sterile water. After that, they were sterilized with 2.5% (
v/
v) NaClO for 8~10 min and rinsed with sterile water. Seeds were inoculated on Murashige and Skoog (MS) medium [
54] hormone-free medium and cultured at 25 °C, 16 h/d light conditions of 4000 lx to obtain sterile seedlings.
4.2. Optimization of Explant Pre-Culture Time
Ten leaf explants were placed on each plate containing callus induction medium (CIM; MS + 1.0 mg·L−1 6-BA + 0.1 mg·L−1 NAA + 3% sucrose + 0.4% phytagel, pH 5.8). A total of six conditions of 2 d, 3 d, 5 d, 7 d, 14 d, and 21 d were established in order to confirm the most suitable pre-culture time and the explants were incubated in the dark at 25 °C. After 4 h of osmotic treatment, the leaf explants were bombarded with the following parameters: 6 cm target distance, 27 mmHg, with 1100 psi rupture disks. The optimal pre-incubation time was evaluated by the frequency of transformed shoots. The methods of bombardment are described in 4.4 and 4.5.
4.3. Selection Pressure of Leaf Explants to Hygromycin B
The concentrations of Hyg were set at five levels of 0, 2.5, 5.0, 7.5, and 10.0 mg·L−1, and the medium was CIM. Leaf explants were placed abaxially and inoculated with each treatment and replicated three times. All the explants were cultured in light at 25 °C for 4 weeks and sub-cultured every 2 weeks. The critical concentration of Hyg was confirmed by counting the induction rate of resistant callus.
4.4. Preparation of Gold Microprojectile
Plasmid PBI-1300 (provided by Pro. Meng Wang, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences) was isolated using the plasmid maxi kit (Omega, United States) according to the manufacturer’s protocol. Transformation conditions were confirmed by plasmid PBI-1300, which harbors the eGFP reporter gene and the hpt selectable gene, both driven by the cauliflower mosaic virus (CaMV) 35S promoter.
The gold particles must be sterilized before being used for DNA coating. An amount of 30 mg of 1.0 μm gold particles were added with 1 mL of 100% ice-cold ethanol and sonicated for 15 s, and were centrifuged at 3000 rpm for 60 s. The supernatant was carefully discarded and the gold particles were resuspended with 1 mL ice-cold ddH2O. The gold particles were centrifuged at 3000 rpm for 60 s and supernatant was removed. The washing step of ddH2O was repeated twice. Finally, the gold particles were suspended with 500 µL of 50% (v/v) sterile glycerin with a final concentration of 60 mg·mL−1. The above microcarriers were stored at −20 °C before use.
The DNA/gold-coating method consists of two levels of optimization (
Table 1). The first method is based on Bio-Rad protocol. An amount of 5 μL DNA (1 μg·μL
−1), 50 μL CaCl
2 (2.5 M), and 20μL Spd (0.1 M) were added to 50 uL microparticle solution, and the mixture was vortexed for 2~3 min and left to stand for 1 min. The supernatant was discarded after centrifugation. The particles were washed with 140 μL of 70% ethanol and absolute ethanol. Finally, the pelleted DNA was suspended with 48 μL of absolute ethanol. The second method corresponded to the previous method [
45]. An amount of 50 μL of gold particles was coated with 10 μL of DNA (1 μg·μL
−1) and supplemented with 10 µL of PM solution (42% PEG 2000 and 560 mM MgCl
2) under vortexing. The mixture was vortexed for 1 min and incubated for 20 min at room temperature. The suspension was centrifuged for 1~5 min. Then, the pelleted DNA was washed with 100 µL of 100% ethanol and was resuspended in 60 µL of 100% ethanol.
4.5. Microprojectile Bombardment
The bombardments were performed with a particle gun (PDS 1000/He, Bio-Rad). The transformation conditions of the particle gun were optimized using one 2-level factor (precipitation agents) and five 3-level factors (target tissue distance, helium pressure, bombardment times, plasmid DNA concentration, and chamber vacuum pressure). These parameters and the levels of the variables studied are shown in
Table 3. A hybrid orthogonal table L
18(2
1 × 3
5) was designed according to the above six parameters and different levels of each parameter [
55], as shown in
Table 6, where A to F represent precipitation agents, target tissue distance, helium pressure, number of bombardments, plasmid DNA concentration, and chamber vacuum pressure. Each factor was repeated three times, and the whole set of experiments was repeated twice.
4.6. Selection and Regeneration of Transformed Plants
The bombarded leaf explants were incubated in the dark at 25 °C for 10~16 h and then transferred to CIM. After 2 days, the transformed leaves were observed for expression of eGFP using Laser Scanning Confocal Microscope (LSM880NLO, Zeiss, German) under an excitation wavelength of 488 nm. The explants were then cut into small pieces of about 1 cm2, placed abaxially on fresh CIM, and cultured at 25 °C, 16 h/d light. After 2 weeks, the transformed cells were observed for the expression of eGFP through LSCM under an excitation wavelength of 488 nm. The explants were then placed abaxially on CIM containing 2.5 mg·L−1 Hyg. After three rounds of selection, the calluses were split into 3~5 pieces and transferred to the regeneration medium (RM; MS + 2.0 mg·L−1 6-BA + 0.2 mg·L−1 NAA + 3% sucrose + 0.4% phytagel, pH 5.8) and incubated at 25 °C under 16 h/d light conditions of 4000 lx. After 2 weeks, green shoots were transferred to shoot elongation medium (SEM; 1/2MS + 2.0 mg·L−1 6-BA + 0.2 mg·L−1 NAA + 3% sucrose + 0.4% phytagel, pH 5.8). For rooting formation, adventitious buds were transferred to root induction medium (RIM; 1/2MS + 0.3 mg·L−1 NAA + 0.5 mg·L−1 IBA + 3% sucrose + 0.4% phytagel, pH 5.8).
4.7. PCR Analysis
The genomic DNA of transformed plants was isolated using CTAB method [
56]. PCR analysis was performed with
hpt (selection maker gene)-specific primers (F: 5′-ATGAAAAAGCCTGAACTCACCG-3′; R: 5′-CTATTTCTTTGCCCTCGGACG-3′) to confirm the transformed strain. The denaturation temperatures used were 98 °C for 30 s, then 35 cycles for amplification with denaturation at 98 °C for 10 s, annealing at 60 °C for 30 s, extension at 72 °C for 20 s, and final extension at 72 °C for 7 min. Then, all the PCR products were sequenced by Sanger sequencing.
4.8. Statistical Analysis
The optimization experiment was repeated three times. The different levels of each experimental factor and the contribution rate of each factor were determined by range analysis and variance analysis. The statistical analysis of all data was calculated by formula. In the range analysis, the values of R and means of Ki were calculated:
The range analysis of the hybrid orthogonal experiment was used to adjust the range R value and compared with the adjusted R’ value, where r is the number of replicates for each level of parameters, and d is the conversion coefficient, which is related to the parameter level:
In the analysis of variance, the sum of squared deviations of the factors was calculated as follows:
In the analysis of variance, if the level of the factor is 2, the sum of squared deviations of the factors was calculated as follows:
The significance of difference was calculated by F-test (* p < 0.05; ** p < 0.01).