阅读说明：本技术 狗尾草遗传保守性高效离体再生方法 (High-efficiency regeneration method of Setaria viridis genetic conservation in vitro ) 是由 朱木兰 孙婧朗 朱新广 郑珂媛 于 2021-08-18 设计创作，主要内容包括：本发明公开一种狗尾草遗传保守性高效离体再生方法,其通过取成熟狗尾草种子消毒后的种子接种至添加2,4-D的MS液体培养基中,液体浅层振荡培养,并得到无菌芽；经过直接不定芽诱导培养和不定芽伸长培养；再经生根培养获得完整的再生植株。本发明从单个芽苗培育至移栽可活的稳定植株,最短仅需35天,能够保持狗尾草的遗传保守性。(The invention discloses a high-efficiency in-vitro regeneration method of Setaria viridis genetic conservation, which comprises the steps of inoculating sterilized seeds of mature Setaria viridis seeds into a MS liquid culture medium added with 2,4-D, carrying out liquid shallow layer oscillation culture, and obtaining sterile buds; performing direct adventitious bud induction culture and adventitious bud elongation culture; then the complete regeneration plant is obtained through rooting culture. The method can keep the genetic conservation of the green bristlegrass in only 35 days from the cultivation of a single bud seedling to the transplantation of a viable stable plant.)

1. A method for the genetic conservative high-efficiency in-vitro regeneration of green bristlegrass herb is characterized by comprising the following steps:

(1) preparation of explant material: inoculating sterilized mature Setaria viridis seeds into MS liquid culture medium containing 1-3 mg/L2,4-D, and performing shaking culture on the liquid shallow layer to obtain sterile buds;

(2) direct adventitious bud induction culture: inoculating the obtained sterile bud in the step (1) to an adventitious bud induction culture medium to generate an adventitious bud; the adventitious bud induction culture medium uses MS as a basic culture medium, and 1-4 mg/L6-BA and 0.1-0.4mg/L NAA are added;

(3) direct adventitious bud elongation culture: inoculating the adventitious bud obtained in the step (2) to an adventitious bud elongation culture medium to obtain an elongated adventitious bud; the adventitious bud elongation culture medium uses MS as a basic culture medium, and 0-0.5 mg/L6-BA and 0-0.09mg/L NAA are added;

(4) rooting culture: dividing cluster buds induced by the adventitious buds in the step (3) into single plants, inoculating the single plants into a rooting culture medium, and performing rooting induction.

2. The method of claim 1, further comprising (5) the steps of hardening off and transplanting.

3. The method according to claim 1, wherein the sterilization method in the step (1) is: treating with 5% potassium permanganate solution for 2min, treating with 75% ethanol solution for 30s, and treating with 10% NaClO solution for 10 min.

4. The method as claimed in claim 1, wherein the culture conditions in steps (3) and (4) are 20-25 ℃, illumination intensity of 2000-.

5. The method according to claim 1, wherein in the step (4), the rooting medium is 1/2MS as a minimal medium, and 1-3mg/L indolebutyric acid is added.

6. The method of claim 2, wherein in the seedling exercising and transplanting step (5), the rooted and strong bottle seedlings in the step (4) are selected for exercising, and the seedling exercising method comprises the following steps: opening the culture bottle cap, injecting 0.5-1cm clear water, standing at room temperature under natural illumination for 2-3 days, taking out bottle seedling, cleaning culture medium attached to the base, and preparing for transplanting.

7. The method of claim 6, wherein said transplanting uses a transplanting substrate of peat soil: perlite: vermiculite 3: 1: 1, soaking the matrix in water to completely wet the matrix before transplanting, bagging and moisturizing after transplanting, spraying the surface of the plant by using a spray can when the humidity is insufficient, removing the bag after the seedling grows sturdy, and maintaining normally.

8. The method of claim 6, wherein the MS medium in steps (2) and (3) and 1/2MS medium in rooting medium are added with sucrose 30g/L, inositol 0.1g/L and agar powder 5.5g/L, and the pH of the medium is adjusted to 5.8.

9. The method according to claim 1, wherein the MS liquid medium in the step (1) is supplemented with sucrose (30 g/L) and inositol (0.1 g/L), and the pH of the medium is adjusted to 5.8.

Technical Field

The invention relates to the field of plant tissue culture, in particular to a method for genetic conservative high-efficiency in-vitro regeneration of green bristlegrass herb.

Background

Setaria viridis (L.) Beauv.) also known as Arohari grass and Echinochloa crusgalli is distributed mainly in temperate, tropical and subtropical regions. Setaria viridis has strong resistance to salt, alkali, drought, and other adverse conditions (Zhang lan Ying, Chen Zhu, Li Gunn. Setaria viridis callus induction and plant regeneration [ J]In the plant physiology communication, 1992(06):433), the yield is high, and meanwhile, the high-quality feed of herbivores (horses, cattle, sheep, and the like) provides guarantee for the development of animal husbandry. Setaria tropical C₄Plants with short growth cycle and small genome (Zhaohui, Zhali, Guo Jing Yu, Hoosmate, Lemna minor, Xiajiyu, Windsor, Guo Anping mannitol stress lower model plant Setaria viridis seed germination stage drought resistance identification and evaluation index research [ J]The school newspaper for tropical crops, 2017,38(11):2060-The method has the advantages of being a model for researching the photosynthesis of the C4 plant and a novel transgenic model plant (Zhaohui, Gujing Yuan, Konhua, Guoaiping, setaria viridis embryonic callus high-efficiency agrobacterium transformation system establishment [ J]The journal of tropical crops, 2017,38(06): 1106-1112). If the excellent characteristics of the green bristlegrass herb can be transferred to grain crops (such as rice), the yield of the grain crops can be increased substantially. The population quantity of the green bristlegrass resistant plants is expanded, and the first step of improving the grain yield is realized. At present, the domestic research on the regeneration of green bristlegrass is rare and few, and mainly focuses on the establishment of a high-efficiency genetic transformation system for transforming green bristlegrass seeds by using agrobacterium tumefaciens assisted by callus induction (Zhaohui, Guo Jing Yuan, Konhua, Guo Anping)]Molecular plant breeding, 2017,15(12): 4955-. Generally, the research on the green bristlegrass tissue culture at home and abroad at present is the regeneration of indirect adventitious buds through callus, and a regeneration system has the defects of genetic instability and long culture time.

In addition, the dormancy phenomenon of the green bristlegrass seeds is serious, so that the germination rate is low, and the improvement of the germination rate of the seeds is also a difficult problem. Studies by Jiafeng et al show that the germination rate of Setaria viridis seeds is highest at 5-15 ℃ and 10-20 ℃ under dark conditions, and can reach 49% and 45%. (Jiafeng, plum blossom, Zhang Hui-Tu, temperature on seed germination of green bristlegrass and Setaria viridis plants [ J]Seed 2016,35(04):30-33+ 43). The alternative temperature change treatment of the Siraitia japonica improves the germination rate of the Setaria viridis seeds to 70.67% (the alternative temperature change treatment of the Siraitia viridis, the King of the Siraitia viridis, the Yangjin Juan, the Lufu Cheng, the Wang He birch, the influence of the low temperature and the temperature change on the germination characteristics of the Setaria viridis seeds [ J]Seed, 2021,40(01): 23-27.). Gibberellins (GA) are used by Rofucheng et al₃) Soaking seeds in solution of indolebutyric acid (IBA), 6-furfurylaminopurine (6-KT) and Ethephon (ETH), wherein the GA concentration is 400mg/L₃The seed soaking effect of the solution is best, and the germination rate is 54%. (Selenocomposition, anethoid, Peng-Jian, Chun-Hui, schwenhua, He-Chao, Guomei-Feng-Gen.) Effect of exogenous hormones on dormancy of Naroxg African Setaria viridis seeds [ J]Grass science 2015,32(03): 406-. 200mg/L GA for use by marjoram et al₃Seed soakingThe treatment has the germination rate of 76 percent, the PEG penetration treatment of 10 percent and the germination rate of 63 percent (Malayaki, Biyufen, Huangmei, Liuqian, fan mei, different gibberellins and the polyethylene glycol treatment have the influence on the gerbera africana seed germination and seedling [ J]The journal of tropical crops, 2009,30(10): 1479-. The highest germination rate of 26 percent (germination test of wild seeds of Chenyanyu, Zenchang Youli, wild oat, golden green bristlegrass, Yunnan gumbo and tarragon [ J ] is achieved by Chenyanyu and the like under the conditions of precooling and KNO3 treatment (20-30 ℃) (J)]A Sichuan stockbreeding veterinarian, 2009,36(10): 31-32.). Guruing et al soaked seeds with 800mg/L GA3 to reach germination rate of 36.67%, and soaked seeds with 25% hydrochloric acid to reach germination rate of 34.00% (Guruing, Zhang Jianhua, Guanwanghui, Wanhuxian, Caochianlin, radix et caulis Opuntiae Dillenii, and research on dormancy breaking method of Setaria viridis seeds [ J ] research [ J]Shanxi agricultural science, 2017,45(07): 1084-. In the previous study, GA was mainly used₃And hormones such as IBA and the like promote the germination of the green bristlegrass herb by a seed soaking treatment mode, and the germination rate is still not high. Therefore, when cultivated by the traditional planting method, the seed germination rate of the green bristlegrass herb is low, and the time consumption is long (ran Guangfu, Wulixian, Chenyongfu, Lujiaxi, Luokejiang, Liu Zu, Xiyanmei, Li jiao, bear Mingbo, Zhouyeju, Chongyi. research and application of the rapid breeding technology of the African green bristlegrass [ J]In the draft, 2020(05) 53-56).

Therefore, a method for researching the genetic conservation and high-efficiency in-vitro regeneration of the green bristlegrass is urgently needed, namely a method for rapidly expanding the number of green bristlegrass populations and ensuring high genetic conservation so as to provide support for transgenic green bristlegrass.

Disclosure of Invention

Aiming at the defects and requirements of the prior art, the invention develops research on dual targets of genetic stability and high propagation coefficient, provides a method for high-efficiency rapid propagation of green bristlegrass, cultivates green bristlegrass seedlings with high genetic stability, and enlarges the population quantity of the green bristlegrass in a short time. The invention provides a high-efficiency adventitious bud in-vitro regeneration method for the first setaria viridis at home and abroad, and provides a most efficient and rapid group propagation method for setaria viridis transgenic resistant strains.

Therefore, the invention provides a high-efficiency regeneration method of Setaria viridis genetic conservation in vitro, which is characterized by comprising the following steps:

(1) preparation of explant material: inoculating sterilized mature Setaria viridis seeds into MS liquid culture medium containing 1-3 mg/L2,4-D, and performing shaking culture on the liquid shallow layer to obtain sterile buds;

(2) direct adventitious bud induction culture: inoculating the obtained sterile bud in the step (1) to an adventitious bud induction culture medium to generate an adventitious bud; the adventitious bud induction culture medium uses MS as a basic culture medium, and 1-4 mg/L6-BA and 0.1-0.4mg/L NAA are added;

(3) direct adventitious bud elongation culture: inoculating the adventitious bud obtained in the step (2) to an adventitious bud elongation culture medium to obtain an elongated adventitious bud; the adventitious bud elongation culture medium uses MS as a basic culture medium, and 0-0.5 mg/L6-BA and 0-0.09mg/L NAA are added;

(4) rooting culture: dividing cluster buds induced by the adventitious buds in the step (3) into single plants, inoculating the single plants into a rooting culture medium, and performing rooting induction;

optionally, the method also comprises (5) the steps of hardening off the seedlings and transplanting.

In a specific embodiment, the sterilization method in the step (1) is: treating with 5% potassium permanganate solution for 2min, treating with 75% ethanol solution for 30s, and treating with 10% NaClO solution for 10 min.

In a preferred mode, the culture conditions in the steps (3) and (4) are 20-25 ℃, the illumination intensity is 2000-.

In the specific embodiment, in the step (4), 1/2MS is used as a minimal medium in the rooting medium, and 1-3mg/L of indolebutyric acid (IBA) is added.

In another embodiment, the operation method of (5) hardening off and transplanting seedlings is as follows: selecting bottle seedlings which have rooted roots and are strong in growth in the step (4) for hardening seedlings, wherein the hardening seedling method comprises the following steps: opening the culture bottle cap, injecting 0.5-1cm clear water, standing at room temperature under natural illumination for 2-3 days, taking out bottle seedling, cleaning culture medium attached to the base, and preparing for transplanting. The transplanting matrix is peat soil: perlite: vermiculite 3: 1: 1, soaking the matrix in water to completely wet the matrix before transplanting, bagging and moisturizing after transplanting, spraying the surface of the plant by using a spray can when the humidity is insufficient, removing the bag after the seedling grows sturdy, and maintaining normally.

Preferably, 30g/L of sucrose, 0.1g/L of inositol and 5.5g/L of agar powder are added into the culture medium in each step, and the pH value of the culture medium is adjusted to 5.8. If the liquid culture medium is not added with agar powder.

The invention has the advantages that: firstly, the research on the green bristlegrass tissue culture at home and abroad is indirect adventitious bud regeneration through callus, and compared with direct adventitious bud regeneration, the green bristlegrass tissue culture has the advantages of genetic instability and long culture time. The method only needs 35 days from the cultivation of a single bud seedling to the transplantation of a viable stable plant, and can keep the genetic conservation of the green bristlegrass. Secondly, the setaria viridis seeds have longer germination time, and the inoculation in a conventional solid culture medium needs about 25 days, the invention firstly proposes that the MS culture medium added with 2,4-D is adopted for carrying out liquid shallow layer shaking culture, so that the germination time is greatly shortened, and only 7 days are needed, thereby the whole time of in-vitro regeneration of the setaria viridis is shortened, and particularly the germination rate is also greatly improved. Thirdly, the research on the rapid propagation technology of green bristlegrass in China is totally focused on the aspect of optimizing the planting technology, and the invention expands the population quantity of the transgenic green bristlegrass by utilizing the tissue culture technology and greatly shortens the breeding time. Fourthly, the invention screens and utilizes the combination of common hormones, obtains the optimal hormone dosage and proportion through research, realizes the high-efficiency in-vitro regeneration of the green bristlegrass while simplifying the hormone types and reducing the cost, and reduces the cost for the research of transferring the excellent characteristics of the green bristlegrass to grain crops. The invention finally achieves the technical scheme of the invention, namely the method for the genetic conservative high-efficiency in-vitro regeneration of the green bristlegrass herb through the aspects.

Drawings

Figure 1 starting explant used in the invention, bar: 1 cm;

FIG. 2 adventitious bud induction, bar: 1 cm;

FIG. 3 adventitious bud induction after removal of apical buds, bar: 1 cm;

FIG. 4 adventitious bud elongation, bar: 0.5 cm;

FIG. 5 adventitious bud rooting, bar: 2 cm;

FIG. 6 soil plant establishment.

Detailed Description

The invention will be better understood by reference to the following description of specific embodiments. But are not to be construed as limiting the invention.

(1) Preparation of explant Material

Taking mature green bristlegrass seeds ME34 as initial explants, carrying out shelling treatment on one part of the mature green bristlegrass seeds, carrying out shelling treatment on the other part of the mature green bristlegrass seeds, firstly using 5 per thousand potassium permanganate solution for 2min, then using 75% ethanol solution for 30s, finally using 10% NaClO solution for 10min, inoculating the disinfected seeds into Murashige & Skoog (MS) liquid culture medium added with 0, 1, 2 and 3mg/L2,4-D, carrying out shaking culture on the liquid shallow layer for 15D, and then carrying out statistics on germination rate. The germinated seeds were inoculated into Murashige & Skoog (MS) solid medium to obtain starting explants (FIG. 1).

The results show that the shelling is more helpful for seed germination, the plant hormone 2,4-D can promote seed germination, and the liquid shallow layer shaking culture can increase the contact area of the seeds and oxygen and supply oxygen fully compared with the conventional solid medium culture. The germination rate is counted at 15 days, the highest germination rate in MS +1 mg/L2,4-D can reach 88.33 percent (table 1), and the germination can be realized in 7 days at the shortest time.

The invention adopts the MS culture medium added with 2,4-D for liquid suspension culture for the first time, greatly improves the germination rate of the green bristlegrass, can reach 88.33 percent, and shortens the germination time at the same time.

TABLE 1 whether shelling and the Effect of different media combinations on seed Germination

(2) Adventitious bud induction culture

Transferring the sterile buds to an adventitious bud induction culture medium, adding 6-BA 1, 2, 3, 4mg/L and NAA 0.1, 0.2, 0.3, 0.4mg/L to the adventitious bud induction culture medium by using MS as a basic culture medium, setting 16 experimental groups and 1 control group in total, and adding 0 mg/L6-BA +0mg/L NAA to the control group. Wherein, 30g/L of sucrose, 0.1g/L of inositol and 5.5g/L of agar powder are added into the MS culture medium, and the pH value of the culture medium is adjusted to 5.8; the culture conditions are 25 +/-2 ℃, the illumination intensity is 2000-. After 25d, statistics: proliferation rate, average number of shoots.

The results show that different concentrations of 6-BA and NAA hormone in combination induce Setaria viridis to produce adventitious buds (Table 1). When the concentration of NAA is constant, the proliferation rate of the adventitious buds and the average bud number show the trend of increasing first and then decreasing along with the increase of the concentration of 6-BA, and the optimal concentration of 6-BA is 2-3 mg/l. When the concentration of 6-BA is constant, the proliferation rate of adventitious buds and the average bud number show the trend of increasing first and then decreasing along with the increase of the concentration of NAA, and the optimum concentration of NAA is 0.2-0.3 mg/l. After 7 days of culture, lateral buds were grown at the stem base (FIG. 2), and adventitious bud induction culture was continued after adventitious bud excision of the seedling. After 25 days, statistics shows that 2mg/l of 6-BA and 0.2mg/l of NAA adventitious bud induction culture medium have the best induction effect, the induction rate reaches 91.11%, the average bud number reaches 49.33, the plant vitality is good, and the adventitious buds are compact (figure 3). In conclusion, the optimal concentration range of 6-BA is 2-3mg/l, the optimal concentration range of NAA is 0.2-0.3mg/l, wherein the optimal hormone combination of 2mg/l of 6-BA and 0.2mg/l of NAA hormone is the optimal hormone combination of the adventitious bud induction culture medium.

TABLE 2 Effect of different phytohormone combinations on adventitious bud Induction

(3) Direct adventitious bud elongation culture

Transferring the adventitious bud in the adventitious bud induction culture to an adventitious bud elongation culture medium. The adventitious bud elongation culture medium uses MS as a basic culture medium, and is respectively added with 0.03, 0.07, 0.1, 0.3 and 0.5mg/L of 6-BA and 0.01, 0.02, 0.03, 0.05, 0.07 and 0.09mg/L of NAA, 25 groups of experimental groups and 1 group of control groups are arranged in total, and 0mg/L of 6-BA +0mg/L of NAA is added to the control group. Wherein, 30g/L of sucrose, 0.1g/L of inositol and 5.5g/L of agar powder are added into the MS culture medium, and the pH value of the culture medium is adjusted to 5.8; the culture conditions are 25 +/-2 ℃, the illumination intensity is 2000-. After 25d, statistics: and (3) elongation.

The result shows that the combination of 26 different hormones of 6-BA and NAA promotes the adventitious bud elongation of green bristlegrass herb, and when the concentration of the NAA is constant, the adventitious bud elongation rate shows the tendency of ascending first and then descending along with the increase of the concentration of the 6-BA. The optimal concentration range of 6-BA is 0.03-0.3mg/L, the optimal concentration range of NAA is 0.01-0.05mg/L, wherein 0.1mg/L of 6-BA +0.03mg/L of NAA is the optimal hormone combination formula for adventitious bud elongation induction culture. Significant elongation of shoots was observed after 15d of culture (FIG. 4).

TABLE 3 Effect of different hormone combinations on adventitious bud elongation

(3) Rooting culture

Dividing cluster buds induced by adventitious buds into individual plants, inoculating the individual plants to a rooting culture medium, and performing rooting induction by using 1/2MS as a basic culture medium and adding 0.1-1mg/L IBA for 7-10 days to root. IBA0.5mg/L is the optimal hormone concentration for adventitious bud rooting, rooting can be realized after 7 days, and the rooting induction rate reaches 98.5%. After 15 days, 25-30 roots were obtained (FIG. 5).

(4) Hardening and transplanting seedlings

Hardening off the rooted and strong bottle seedlings, wherein the hardening off method comprises the following steps: opening the culture bottle cap, injecting 0.5-1cm clear water, standing at room temperature under natural illumination for 2-3 days, taking out bottle seedling, cleaning culture medium attached to the base, and preparing for transplanting. The transplanting matrix is peat soil: perlite: vermiculite 3: 1: 1, soaking the substrate in water before transplanting to completely wet the substrate, bagging and moisturizing after transplanting, spraying the substrate on the surface of a plant by using a spray can when the humidity is insufficient, removing the bag after the seedling grows sturdy, and normally maintaining (figure 6). And after 1 month, the survival rate is counted and is over 95 percent.