Method for carrying out herbicide-resistant germplasm innovation by mutagenizing salvia miltiorrhiza callus through EMS

Abstract

The invention discloses a method for carrying out herbicide-resistant germplasm innovation by mutagenizing salvia miltiorrhiza calluses through EMS. The method comprises the following steps: carrying out mutagenesis treatment on salvia miltiorrhiza calluses by using EMS under proper treatment conditions; under the 2 times of field recommended metering of glyphosate spraying, the tolerance of the salvia miltiorrhiza seedlings treated by EMS and screened by glyphosate is obviously enhanced, and the glyphosate-resistant enhanced salvia miltiorrhiza germplasm is obtained. In order to facilitate later mutant genotype identification, salvia miltiorrhiza calluses with consistent genotypes are selected for EMS mutagenesis, salvia miltiorrhiza glyphosate critical dose screening is carried out for the first time, glyphosate-resistant salvia miltiorrhiza germplasm is successfully screened by virtue of a mature salvia miltiorrhiza tissue culture rapid propagation technology, and a set of effective EMS mutagenesis method is provided for cultivating excellent salvia miltiorrhiza varieties.

Description

Technical Field

[0001] The invention relates to the field of mutation breeding, and in particular to a method for herbicide-resistant germplasm innovation by utilizing EMS to induce mutation of salvia miltiorrhiza callus. Background Art

[0002] Ethyl methanesulfonate (EMS) mutagen is an alkylating agent and is most frequently used in chemical mutagenesis breeding. Most mutations induced by EMS are dominant, and the frequency of point mutations is relatively high. The number of chromosome aberrations during the mutagenesis process is relatively small, which can improve the special germplasm traits of plants more specifically, such as herbicide resistance and drought resistance. Therefore, it is widely used in the construction of plant mutant libraries and germplasm resource innovation.

[0003] Salvia miltiorrhiza is a perennial herb of the genus Salvia in the family Lamiaceae. The dried roots and rhizomes of Salvia miltiorrhiza are one of the commonly used bulk medicinal materials in my country (China Pharmacopoeia Committee, 2020). Salvia miltiorrhiza has a wide range of uses and significant therapeutic effects. It has the effects of promoting blood circulation and removing blood stasis, anti-tumor, anti-oxidation, and anti-inflammatory. In the cultivation process of Salvia miltiorrhiza, weeds are an important factor affecting quality, economic benefits and yield. They compete with crops for resources such as water, light and nutrients, resulting in a decrease in Salvia miltiorrhiza yield. Traditional manual weeding is time-consuming and labor-intensive, and it is not timely and ineffective for large-scale control. The use of chemical herbicides has improved the control efficiency of weeds in farmland, but there is currently no special herbicide for Salvia miltiorrhiza, and there are strict regulations on the use of pesticides in the cultivation of Chinese medicinal materials. Cultivating Salvia miltiorrhiza varieties that are resistant or tolerant to herbicides is one of the solutions. At present, no relevant results have been reported in China.

[0004] Glyphosate is a systemic stem-and-leaf herbicide with broad-spectrum lethality and high efficiency. Studies have shown that glyphosate quickly loses its effect after entering the soil and is degraded by soil microorganisms. The final products of degradation are water, CO2 and phosphoric acid, and the degradation products are not absorbed by plants.

[0005] There are currently no varieties of Salvia miltiorrhiza that can be treated with glyphosate for weed control. Summary of the invention

[0006] In the existing cultivation process of Salvia miltiorrhiza, there is a lack of technology for efficiently cultivating herbicide-resistant Salvia miltiorrhiza, and the efficiency is low at the callus emergence stage, which limits the variety improvement and large-scale planting of Salvia miltiorrhiza. The present invention aims to solve the above problems and provide a comprehensive cultivation and optimization technology to improve the herbicide resistance and cultivation efficiency of Salvia miltiorrhiza.

[0007] The technical solution of the present invention mainly includes the following aspects:

[0008] Cultivation of herbicide-resistant Salvia miltiorrhiza callus: The leaves of a specific Salvia miltiorrhiza variety (such as Shannong Salvia miltiorrhiza No. 1) are used as the starting material, and callus formation is induced after disinfection. The callus is treated with EMS solutions of different concentrations (prepared with phosphate buffer as the solvent), and multiple concentration gradients and treatment time combinations are set. By comparing the survival rate of callus under different treatments, the half-lethal dose of EMS-induced Salvia miltiorrhiza callus is determined. The callus treated with a half-lethal dose of EMS is propagated and cultured, and then the herbicide-resistant callus is screened on a culture medium containing a critical dose of herbicide. After multiple subcultures, stable herbicide-resistant Salvia miltiorrhiza callus is obtained.

[0009] Optimization methods for improving the germination rate of Salvia miltiorrhiza callus: In response to the problems of slow germination and low germination rate of callus after EMS mutagenesis treatment, a variety of optimization methods have been invented. The first is low-temperature treatment. After the callus is cut into small pieces and inoculated into a specific solid budding medium, it is treated with low temperature (such as 4°C) for different lengths of time, and then cultured at room temperature. The appropriate low-temperature treatment duration is determined through experiments to promote germination. The second is liquid oscillation culture treatment. Small pieces of callus are inoculated into a liquid culture medium containing specific ingredients, and dark oscillation culture is carried out. Different culture times are set, and after culture, it is transferred to a solid culture medium. Studies have found that the appropriate liquid oscillation culture time can significantly improve the germination rate. The third is to adjust the composition of the culture medium, change cytokinins and other components, compare the germination of callus under different culture medium compositions, and screen out a culture medium formula that can make the callus sprout earlier and with a higher germination rate. By combining these optimization methods, the germination rate of callus tissue has been greatly improved.

[0010] Screening and identification of herbicide-resistant Salvia miltiorrhiza plants: Transplant Salvia miltiorrhiza seedlings treated with a semi-lethal dose of EMS, select mutant plants with consistent growth and spray them with a specific concentration (such as 2 times the recommended field dose) of the target herbicide (such as glyphosate) in groups, and use Salvia miltiorrhiza plants that have not been treated with EMS mutagenesis and sprayed with the same herbicide as a control. At a specific time after spraying the herbicide (such as the 7th day), the plant's phytotoxicity is judged according to the established phytotoxicity grade standard, and Salvia miltiorrhiza plants with herbicide-resistant characteristics are screened out, thereby completing the screening and identification process of herbicide-resistant Salvia miltiorrhiza plants.

[0011] Through the above technical scheme, the present invention has achieved remarkable beneficial effects: successfully cultivated herbicide-resistant Salvia miltiorrhiza callus and plants, improved the tolerance of Salvia miltiorrhiza to herbicides, provided protection for the growth of Salvia miltiorrhiza in agricultural production environments using herbicides, reduced weed competition, and improved the yield and quality of Salvia miltiorrhiza; optimized the germination conditions of Salvia miltiorrhiza callus, greatly improved the germination rate, accelerated the reproduction speed of Salvia miltiorrhiza, shortened the cultivation cycle, improved the cultivation efficiency of Salvia miltiorrhiza, and was conducive to the rapid promotion and application of excellent varieties of Salvia miltiorrhiza. . BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 To obtain herbicide-resistant Salvia miltiorrhiza callus.

[0013] Figure 2 This is the callus budding after 4℃ low-temperature dark culture, from left to right: CK, low-temperature treatment for 5h, 10h, and 24h.

[0014] Figure 3 This is the callus budding after 1-6 days of low-speed liquid shaking culture, from left to right: CK, liquid culture 1d, 2d, 3d, 6d.

[0015] Figure 4 This is the culture of Salvia miltiorrhiza callus induced by EMS solution.

[0016] Figure 5 The leaf shape and edge notch characteristics, among which A is elliptical and serrated; B is oval and rounded; C is nearly circular and serrated. DETAILED DESCRIPTION

[0017] Example 1: Shannong Danshen No. 1 is used as the culture material.

[0018] 1 Materials and methods

[0019] 1.1 Test materials

[0020] The Salvia miltiorrhiza callus used in this experiment was cultured with Shannong Salvia miltiorrhiza No. 1. Its leaves were sterilized with mercuric chloride and 75% alcohol and cut into 1 cm 2 Callus tissue cultured from small pieces of tissue.

[0021] 1.2 Test methods

[0022] 1.2.1 Preparation of EMS solution

[0023] Using phosphate buffer as solvent, EMS solutions with concentrations of 0, 0.4%, 0.6%, 0.8%, 1.0%, 1.5% and 2% were prepared respectively.

[0024] 1.2.2 EMS solution induced mutation of Salvia miltiorrhiza callus

[0025] The test of inducing mutation of Salvia miltiorrhiza callus was carried out on a clean bench, and 6 concentration gradients (0.4%, 0.6%, 0.8%, 1.0%, 1.5%, 2.0%) of EMS solution were set, and phosphate buffer without EMS was used as the control (CK). On the clean bench, the callus was cut into small pieces of about 3 mm in size, soaked in solutions of different concentrations for 4, 6, and 8 hours, rinsed with sterile water 5-6 times, and dried with sterile filter paper, and inoculated on the callus subculture medium. There were 10 callus in each bottle of culture medium, 3 bottles of culture medium for 1 treatment, and each treatment was repeated 3 times. Then it was placed in an intelligent light incubator at a temperature of 26°C and the callus survival rate was observed after two weeks, and the optimal half-lethal dose of EMS for Salvia miltiorrhiza callus was finally determined.

[0026] Callus survival rate (%) = number of surviving calli / total number of calli × 100;

[0027] Relative callus survival rate (%)=(callus survival rate of each EMS mutagenesis treatment combination / callus rate of the control group)×100.

[0028] Lethal concentration 50 (LC50): determined based on the relative survival rate of callus tissue treated with EMS.

[0029] 1.2.3 Identification of the toxicity of herbicides to Salvia miltiorrhiza callus

[0030] Select callus tissues with emerald green color, compact texture and vigorous growth for toxicity identification, cut them into callus tissue blocks of uniform size and inoculate them on callus subculture medium with five doses of herbicide added at one, two, four, six and eight times the recommended concentration. Use callus subculture medium without herbicide as control, inoculate 10 callus tissues in each bottle, 3 bottles of culture medium as one treatment, and repeat 3 times for each treatment. Place in a 25℃ culture room and culture with 15h / d light. Observe and record the proliferation and browning death of callus tissue after one month to determine the lethal dose and critical dose of herbicide to callus.

[0031] 1.2.4 Screening of herbicide-resistant Salvia miltiorrhiza calli

[0032] (1) Screening of herbicide-resistant Salvia miltiorrhiza calli

[0033] The callus tissue treated with a semi-lethal dose of EMS solution was propagated and cultured. The surviving callus tissue was subcultured 3 times (once every 7-10 days), and then inoculated on a culture medium containing a critical dose of herbicide for screening. It was subcultured every 20 days, and after being transferred 2-3 times, it was transferred to a normal culture medium without herbicide. The surviving callus tissue obtained was the screened mutant that may be resistant to herbicides.

[0034] (2) Cultivation of Salvia miltiorrhiza callus induced by EMS solution

[0035] The callus tissues that survived the semi-lethal dose of EMS solution were redifferentiated and cultured until they became seedlings, and then planted in flower pots for further cultivation.

[0036] 1.2.5 Optimization test of callus emergence

[0037] In order to accelerate the differentiation of adventitious buds from callus tissue and increase the proportion and reproduction coefficient of differentiated adventitious buds, three small experiments were designed:

[0038] 1) Low temperature treatment: Cut the callus into pieces of about 0.5 cm 3 The small pieces were inoculated into solid budding medium and subjected to short-term low-temperature treatment at 4°C for 5h, 10h and 24h respectively. The callus tissue that was not subjected to low-temperature treatment was used as the control. Each treatment was repeated 3 bottles. After low-temperature treatment, the callus was placed in a normal temperature incubator for culture. The medium composition was MS+2.0mg / L6-BA+0.5mg / L NAA, with 10 grains placed in each bottle.

[0039] 2) Liquid shaking culture treatment: Cut the callus into pieces of about 0.5 cm 3 The small pieces were inoculated into the liquid culture medium, the composition of which was MS+2.0mg / L 6-BA+0.5mg / L NAA+1g / L PVP. The culture medium without PVP was used as the control. A 50ml triangular flask was added with 30ml of culture medium. After inoculation, it was cultured in dark shaking for 1d, 2d, 3d, and 6d, respectively, at a speed of 80r / min, and each treatment was repeated for 3 bottles. After the liquid culture treatment, solid culture medium was inoculated, and the culture medium composition was MS+2.0mg / L 6-BA+0.5mg / L NAA.

[0040] 3) Adjustment of culture medium composition: The culture medium composition was adjusted to MS+2.0mg / L KT+0.5mg / L NAA, and the original culture medium composition MS+2.0mg / L 6-BA+0.5mg / L NAA was used as the control. Eight grains were inoculated into each bottle, and each treatment was repeated for 3 bottles.

[0041] The cells were cultured under 15 h / d light, and the differentiation of adventitious buds from callus was observed and recorded for 7 days. The callus blocks with adventitious buds were counted and the budding rate was calculated.

[0042] 1.2.5 Investigation of traits of EMS-induced plant populations

[0043] The characteristics of EMS-induced Salvia miltiorrhiza population were investigated at the seedling stage, mainly focusing on leaf color, leaf shape and leaf edge, and the mutant phenotypes were statistically screened and recorded.

[0044] 1.2.6 Identification of herbicide-resistant individual plants

[0045] The seedlings of Salvia miltiorrhiza were treated with a semi-lethal dose of EMS solution and then transplanted. After transplanting, the mutant plants of Salvia miltiorrhiza were screened for herbicide resistance. The materials with the same growth were selected and grouped to spray the target herbicide glyphosate, and the spraying concentration was designed to be 2 times the recommended field dosage (4500g / hm2). Shannong Salvia miltiorrhiza No. 1, which was sprayed with glyphosate but not treated with EMS, was used as a control. Five replicates were set for each treatment, and the experimental effects were observed on the 7th day after treatment. The phytotoxicity was judged according to the glyphosate injury grade standard (HIS), and the Salvia miltiorrhiza EMS mutant plants with herbicide resistance were preliminarily screened out.

[0046] Table 1 Glyphosate toxicity level standards

[0047]

[0048] 2 Results and analysis

[0049] 2.1 Effects of EMS mutagenesis combinations on Salvia miltiorrhiza callus

[0050] After 1 day of culture, part of the Salvia miltiorrhiza callus treated with EMS solution began to turn yellow. After 3 days of culture, part of the callus turned brown. As the culture time increased, the browning gradually deepened, and some materials finally died. The surviving materials were yellow-green or emerald green. After 14 days of culture, the survival rate was counted (see Table 1). It can be seen from the table that the survival rate of Salvia miltiorrhiza callus treated with EMS was lower than that of the control, indicating that EMS has a toxic effect on Salvia miltiorrhiza callus. And as the EMS concentration increased, the survival rate decreased significantly. All Salvia miltiorrhiza callus treated with 2% EMS solution died, indicating that this concentration had the greatest toxic effect on Salvia miltiorrhiza callus. When the mutagenesis time was 4h and the EMS concentration was 1%, the survival rate of Salvia miltiorrhiza callus was 56.7%. The final half-lethal dose was determined to be 1% and 4h, which was the optimal treatment condition for EMS mutagenesis of Salvia miltiorrhiza callus.

[0051] Table 1 Effects of different EMS concentrations and treatment time on callus survival rate

[0052]

[0053] 2.2 Investigation of glyphosate toxicity to Salvia miltiorrhiza callus

[0054] As shown in Table 2, with the increase of glyphosate treatment concentration, the browning rate of Salvia miltiorrhiza callus gradually increased and the proliferation rate gradually decreased. When the glyphosate concentration was 0.15g / L, its proliferation rate was no different from the control; when the glyphosate concentration was 0.3g / L, the proliferation rate of Salvia miltiorrhiza callus decreased to 40%, which was significantly different from the control group and the 0.15g / L glyphosate treatment group, and the Salvia miltiorrhiza tissue browned significantly; under the glyphosate concentration of 0.375g / L, most of the Salvia miltiorrhiza callus browned and died, and the proliferation rate was only 23.33%, which was suitable for glyphosate-resistant mutant screening; under the glyphosate concentration of 0.60g / L, the browning rate was 100%, and all the Salvia miltiorrhiza callus died. Therefore, its lethal concentration is 0.60g / L and the critical concentration is 0.375g / L.

[0055] Table 2 Effects of different concentrations of glyphosate on callus growth

[0056]

[0057] 2.3 Screening of herbicide-resistant Salvia miltiorrhiza callus

[0058] Select callus tissues with emerald green color, compact texture and vigorous growth, treat them with 1% EMS solution for 4 hours, and then inoculate them on callus subculture medium. After subculture three times, the surviving callus is inoculated on callus medium containing 0.375g / L glyphosate to screen herbicide-resistant mutants. After repeated screening for three generations, the physiologically adaptable Salvia miltiorrhiza callus tissues are removed, and the surviving callus tissues are herbicide-resistant variants, which are propagated to obtain glyphosate-resistant callus. Figure 1 As shown in C and D.

[0059] 2.4 EMS-induced callus emergence of Salvia miltiorrhiza

[0060] The callus tissue treated with EMS grew faster, but the differentiation rate of adventitious buds was significantly slower than that of the control material, and the number of adventitious buds that could be induced was very small. To this end, we designed a variety of methods such as low temperature stimulation, solid-to-liquid shaking culture, and optimized culture group composition, which significantly improved the germination rate. Specific optimization measures include:

[0061] 2.4.1 Low temperature treatment

[0062] The experiment was designed to treat the plants in the dark for 5h, 10h, and 24h at 4℃, and then to continue growing under the conditions of 25℃ temperature and light. The study found that after 24h 4℃ low temperature treatment, about 15% of the calli could induce adventitious buds, and there was no significant difference in the germination rate under the treatment of 5h and 10h ( Figure 2 ).

[0063] 2.4.2 Low-speed liquid shaking culture

[0064] After 1-3 days of liquid shaking culture, the callus was transferred to solid culture medium. The study found that after 2-3 days of liquid culture, and then 7-14 days of solid culture medium, the budding rate of callus tissue was significantly improved, and about 30% of the callus induced adventitious buds ( Figure 3 ).

[0065] 2.4.3 Optimization of culture medium composition

[0066] When the culture composition of MS+1.5mg / L BA+0.2mg / L NAA was changed to MS+2.0mg / L KT+0.2mg / L NAA+PVP protective agent, the budding of callus tissue was significantly earlier than that of the control (original culture medium), and the budding rate was also significantly improved.

[0067] By optimizing the culture conditions (5-24 hours of 4°C low temperature treatment + low-speed (80-100r / min) liquid culture medium (MS+1.5mg / L BA+0.2mg / L NAA) shaking culture (1-3 days) + transfer to solid culture medium), and adjusting the composition of the seedling medium ((MS+1.5mg / L 6-BA+0.2mg / L NAA replaced by (MS+2.0mg / L KT+0.2mg / L NAA+PVP protective agent), finally 60% of the calluses can induce adventitious buds and transform into rooting medium, and nearly 80 tissue culture seedlings are cultivated.

[0068] 2.5 Phenotypic identification

[0069] Individual plant type variations were found during the tissue culture seedling stage, such as Figure 4 The control material on the left is Shannongdan No. 1, and 1 and 2 are Salvia miltiorrhiza seedlings cultivated from EMS-induced callus. Compared with the control, the induced material has more stems and longer stem nodes, and the individual plants obtained after the expansion of the variant material also have this kind of mutation.

[0070] At the same time, the 80 tissue culture seedlings obtained were identified for morphology, and it was found that there were great differences in leaf shape, leaf color, edge notches and seedling growth. Among them, 70% of the plants had light green leaves in the seedling stage, 25% had medium green leaves, and 5% had dark green leaves. The leaves were mostly oval and nearly round, with a few being elliptical. Among them, 38% of the plants were oval, 48% were nearly round, and the least number of plants were elliptical, accounting for 14% ( Figure 5 ) Regarding leaf margin notches, blunt serrations accounted for 46% of the total number, while round serrations accounted for 54% of the total number.

[0071] 2.6 Identification of herbicide-resistant individual plants

[0072] Seven days after spraying 4500g / hm2 of glyphosate, the salvia miltiorrhiza in the control group fell over and its leaves rolled up, showing obvious signs of damage. The plants dried up and died. According to the glyphosate critical grade standard, almost all plants reached grade five.

[0073] After seven days of treatment with 4500g / hm2 of glyphosate, the EMS-induced material showed leaf chlorosis and leaf edge spot phenotypes, but most of the Salvia miltiorrhiza seedlings survived, and were in the first and second levels of glyphosate criticality, and the newly grown young leaves were normal and healthy. EMS-induced tissue culture seedlings improved the resistance of Salvia miltiorrhiza to glyphosate, allowing Salvia miltiorrhiza seedlings to survive when sprayed with glyphosate at 2 times the recommended field dose.

[0074] The above is only a preferred implementation of this patent. It should be pointed out that for ordinary technicians in this technical field, several improvements and substitutions can be made without departing from the technical principles of this patent. These improvements and substitutions should also be regarded as the scope of protection of this patent.

Claims

1. A method for herbicide-resistant germplasm innovation using EMS-induced salvia miltiorrhiza callus, characterized in that: The following steps are involved: ST1. EMS mutagenesis treatment of Salvia miltiorrhiza callus; ST2. Screening of herbicide-resistant Salvia miltiorrhiza callus; ST3. Low temperature treatment, low-speed liquid shaking culture, and optimization of culture medium composition promote the emergence of callus of induced Salvia miltiorrhiza; ST4. Phenotypic identification, identification of mutant Salvia miltiorrhiza plants; ST5. Identification of individual plant resistance to herbicides.

2. The method for herbicide-resistant germplasm innovation using EMS-induced salvia miltiorrhiza callus according to claim 1, characterized in that: The ST1.EMS mutagenesis treatment of Salvia miltiorrhiza callus has an EMS concentration of 1% and a mutagenesis time of 4 hours.

3. The method for herbicide-resistant germplasm innovation using EMS-induced salvia miltiorrhiza callus according to claim 1, characterized in that: The ST2. screening of herbicide-resistant Salvia miltiorrhiza callus, and screening of glyphosate-resistant mutants under a glyphosate concentration of 0.375 g / L.

4. The method for herbicide-resistant germplasm innovation using EMS-induced salvia miltiorrhiza callus according to claim 1, characterized in that: Among them, ST3. low temperature treatment, low-speed liquid shaking culture, and optimization of culture medium composition promote the emergence of induced Salvia miltiorrhiza callus. The low temperature treatment is 24 hours of 4°C low temperature treatment to induce adventitious buds.

5. The method for herbicide-resistant germplasm innovation using EMS-induced salvia miltiorrhiza callus according to claim 1, characterized in that: Among them, ST3. low temperature treatment, low-speed liquid shaking culture, and optimization of culture medium composition promote the emergence of induced Salvia miltiorrhiza callus. The low-speed liquid shaking culture is to transfer from 2-3 days of liquid shaking culture to solid culture medium for 7-14 days of culture to induce adventitious buds.

6. The method for herbicide-resistant germplasm innovation using EMS-induced salvia miltiorrhiza callus according to claim 1, characterized in that: Among them, ST3. low temperature treatment, low-speed liquid shaking culture, and optimization of culture medium composition promoted the emergence of induced Salvia miltiorrhiza callus. The culture medium composition was optimized by changing the culture composition MS+1.5mg / L BA+0.2mg / L NAA to MS+2.0mg / L KT+0.2mg / L NAA+PVP protective agent to improve the germination rate.

7. The method for herbicide-resistant germplasm innovation using EMS-induced salvia miltiorrhiza callus according to claim 1, characterized in that: The ST4. phenotypic identification is to determine the mutant Salvia miltiorrhiza plants, investigate the leaf color, leaf shape, and edge of Salvia miltiorrhiza, and statistically screen and record the mutant phenotypes.

8. The method for herbicide-resistant germplasm innovation using EMS-induced salvia miltiorrhiza callus according to claim 1, characterized in that: The ST5. herbicide-resistant single plant resistance identification is to screen Salvia miltiorrhiza EMS mutant plants with herbicide resistance characteristics after being treated with glyphosate at a concentration of 4500g / hm2 for seven days.

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