A widely applicable and efficient peanut somatic embryo induction and transformation method

Abstract

The present invention belongs to the field of plant tissue culture technology, and in particular to a wide and efficient peanut somatic embryo induction and transformation method. The present invention first inoculates peanut seeds in MS culture medium for pre-culture, then removes cotyledons and leaves embryos, and slices the connection between embryo leaflets and embryo axes with a blade (cuts 3-4 thin slices), then the thin slices are placed in a dedifferentiation culture medium and cultured until the thin slices become transparent, and then the embryogenic cells are proliferated, and then the embryogenic tissue is obtained and then somatic embryo induction and subculture (or resistance screening) are performed under light, and finally the target somatic embryo is placed in light for germination and regeneration culture, and peanut somatic embryos can be obtained. A large number of healthy somatic embryos can be obtained in a short period of time by the method of the present invention, and the technology is stable, and each peanut variety has universality, and has a huge application prospect in the genetic improvement of peanuts.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and in particular relates to a widely applicable and efficient peanut somatic embryo induction and transformation method. Background Art

[0002] Peanut (Arachis hypogaea L.) is an important economic crop for both oil and food. Cultivating high-yield, high-quality and specialized peanuts is an important goal of peanut breeding. However, due to the complex genome of tetraploid cultivated peanuts, traditional peanut breeding is time-consuming and inefficient, making it difficult to achieve breakthrough progress. With the development of molecular biology, the establishment and optimization of molecular breeding technology will help to explain the genetic function of peanuts, solve the difficulties of traditional peanut breeding, and is an important means of genetic improvement of peanut varieties in the future.

[0003] Genetic transformation of plants is a prerequisite for molecular breeding. At present, the main methods for genetic transformation of plants include Agrobacterium, gene gun-mediated and virus infection, and gene introduction can be achieved through tissue culture and non-tissue culture methods. For example, the applicant previously established a rapid genetic transformation method for peanuts through non-tissue culture methods, which greatly improved the transformation efficiency and shortened the time for genetic material construction, but the vast majority of the offspring obtained were chimeras, with only a few stable genetic offspring [Zhai Qiong, Chen Rongqin, Liang Xiaohua, et al. Establishment and application of a rapid genetic transformation method for peanuts. Acta Botanica Sinica, 2022, 57(03)]. After nearly 10 years of exploration, the peanut tissue culture system has become increasingly mature. Most of the explants are embryo leaflets, hypocotyls, leaves or cotyledon nodes for induction of adventitious buds or somatic embryos. However, there are still problems such as low efficiency, large differences in the induction of different genotypes of peanut varieties, and imperfect seedling systems [Wang Wei, He Zhongguo, Niu Hailong, et al. Research progress in peanut tissue culture and genetic transformation. Molecular Plant Breeding. 2022, 20(23); Qiao Lixian, Sui Jiongming, Tan Lingling, et al. Peanut somatic embryo induction and plant regeneration. Acta Agronomica Sinica. 2012, 2(10); Li Wenjing. Optimization of peanut regeneration system and transformation of AhSAD gene promoter. Master's thesis of Zhengzhou University. 2016.]. Therefore, establishing a complete, efficient and widely adaptable regeneration and genetic transformation system remains the research focus of peanut genetic engineering breeding.

[0004] Plant tissue culture regeneration is divided into two pathways: organogenesis and somatic embryogenesis. Organ regeneration is further divided into direct organ regeneration and indirect organ regeneration. The former does not form callus through dedifferentiation, but can directly induce adventitious buds (clustered buds) from stem tips, peanut buds, etc. to form complete plants. This method is simple and rapid, but is obviously restricted by genotypes, and because adventitious buds occur in multiple cells, the resulting transformed plants are all chimeras. The indirect organ regeneration pathway is a process in which mature cells undergo dedifferentiation and redifferentiation to form new tissues and organs. For example, by inducing embryonic leaflets, leaves, hypocotyls, etc. to form callus, then transforming and inducing adventitious buds, theoretically, non-chimera transformed plants can be obtained with a certain probability, but this pathway also has problems such as low peanut regeneration frequency, large genotype differences, slow bud growth, and difficulty in root induction. Somatic embryogenesis refers to the morphological process of a diploid or haploid somatic cell developing a new individual under specific conditions without sex cell fusion, in a similar way to zygotic embryogenesis. The structure similar to the zygotic embryo formed through somatic embryogenesis is called an embryoid or somatic embryo. Somatic embryos are mostly generated from a single embryogenic cell, which can greatly reduce the probability of generating chimeras. There are many ways to induce somatic embryos in different species or the same species, and there are also many factors that affect the growth of somatic embryos, such as hormone types and ratios, explant sources and developmental stages, varieties and genotypes, osmotic agents and sucrose, activated carbon and light, etc. These factors will affect the quantity and quality of somatic embryos. At present, the main way to generate peanut somatic embryos is to use embryo leaflets, epicotyls, immature embryos and young leaves as explants, and then use hormones such as 6BAP, NAA, PYM (pingyangmycin), 2,4-D to induce different varieties of peanuts to produce somatic embryos. The induction rate ranges from 7.5% to 92%, but there are still problems such as low embryo formation rate, low regeneration of normal plants, and poor repeatability. Therefore, it is necessary to develop a stable and efficient method for inducing peanut somatic embryos. Summary of the invention

[0005] In order to overcome the deficiencies of the above-mentioned prior art, the present invention proposes a widely applicable and efficient peanut somatic embryo induction method. The method of the present invention can obtain a large number of healthy somatic embryos in a short period of time, and the technology is stable and applicable to various varieties of peanuts, and has great application prospects in the genetic improvement of peanuts.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] The present invention provides a widely applicable and efficient peanut somatic embryo induction and transformation method, which comprises the following steps:

[0008] S1. After pre-culturing peanut seeds, two cotyledons are removed to leave young embryos, 3 to 4 thin slices are cut from top to bottom at the junction of the embryo leaflet and the embryo axis, and the cut slices are cultured in a dedifferentiation medium until the slices become transparent, wherein the dedifferentiation medium includes MS, 2,4-D (2,4-dichlorophenoxyacetic acid), pic (picloram), 6-BAP (6-benzylaminopurine) and sucrose;

[0009] S2, converting the dedifferentiated transparent thin slices to an embryonic callus induction medium, and culturing until the transparent thin slices grow many sand-like embryogenic cells, wherein the embryonic callus induction medium comprises MS, 2,4-D, Pic, ZT (zeatin), ser (serine), gln (glutamine) and sucrose;

[0010] S3, transferring the transparent sand-like embryonic callus to a somatic embryo induction and subculture medium for culture until a large number of somatic embryos grow out, wherein the somatic embryo induction and subculture medium comprises MS, Pic, ZT, activated carbon and sucrose;

[0011] S4. Transferring the somatic embryos to a germination medium and growing them under light, and replacing them with a growth medium for seedling culture after the tops of the somatic embryos begin to turn green, wherein the germination medium comprises MS, activated carbon and sucrose, and the growth medium comprises MS, activated carbon, 6-BAP and sucrose.

[0012] Preferably, the varieties of peanuts include Zhanyou 65, Zhanyou 75, Yueyou 7, Yueyou 13, Guihua 21, Fuhua 9 and Zhonghua 16.

[0013] Preferably, the dedifferentiation medium comprises MS, 5 mg / L 2,4-D, 5 mg / L pic, 0.5 mg / L BAP and 3% sucrose. The combined use of 2,4-D and pic as dedifferentiation auxins greatly reduces the browning of explants in the later stage compared to the use of high-concentration single 2,4-D, and basically every slice can induce somatic embryos, which can greatly improve the induction rate of somatic embryos.

[0014] Preferably, the embryonic callus induction medium includes 1 / 2MS, 5mg / L 2,4D, 3mg / L Pic, 0.2mg / LZT, 0.05-0.1g / L ser, 0.3-0.5g / L gln and 2% sucrose. The use of an appropriate amount of serine can eliminate the genotype differences between most peanut varieties. The above culture medium can well induce somatic embryos. Generally speaking, the thicker the pods, the higher the serine concentration required. At the same time, under the premise of pic, the use of low concentrations of zeatin and glutamine can promote the proliferation of embryogenic cells and increase the amount of somatic embryos formed.

[0015] Preferably, the somatic embryo induction and subculture medium comprises 1 / 2MS, 3 mg / L Pic, 0.2 mg / L ZT, 0.1% activated carbon and 2% sucrose.

[0016] Preferably, the germination medium comprises 1 / 2MS, 0.1% activated carbon and 1% sucrose.

[0017] Preferably, the growth medium comprises 1 / 2 MS, 0.1% activated carbon, 4 mg / L 6BAP and 1% sucrose.

[0018] Preferably, the thickness of the sheet in S1 is 1 to 3 mm.

[0019] Preferably, the peanut seed pre-culture in S1 is to culture the peanut seeds in MS medium for 4-6 days.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention discloses a widely applicable and efficient peanut somatic embryo induction method, wherein peanut seeds are first inoculated in MS culture medium for pre-culture, and then the cotyledons are removed to retain the embryos, and the connection between the embryo leaflets and the embryo axis is sliced ​​with a blade (cut into 3-4 thin slices), and then the thin slices are placed in a dedifferentiation culture medium (MS+2,4-D+pic+BAP+sucrose) and cultured until the thin slices become transparent, and then embryogenic cells are proliferated (MS+2,4-D+pic+ZT+ser+gln+sucrose), and after obtaining embryogenic tissue, somatic embryo induction and subculture (or resistance screening) (MS+pic+activated carbon) are performed in the dark, and finally the target somatic embryos are placed in light for germination (MS+activated carbon+sucrose) and regeneration culture (MS+activated carbon+6BAP+sucrose), and peanut somatic embryos can be obtained. In general, the present invention has the following advantages:

[0022] (1) Previous studies have mostly used embryonic leaflets to induce somatic embryos, and the number of explants and somatic embryos obtained is limited. However, the present invention cuts the connection between the peanut embryonic leaflet and the hypocotyl into thin slices, which not only increases the number of explants, but also makes the thin slices fully dedifferentiated, making it easier to induce embryogenic cells.

[0023] (2) By combining 2,4-D and Pic for dedifferentiation induction, the browning of explants was greatly reduced compared with the use of high concentration 2,4-D (10 mg / L) alone, and somatic embryos could be induced in almost every slice;

[0024] (3) Before somatic embryo induction, low concentrations of zeatin (ZT) and glutamine (gln) were used to proliferate embryogenic cells, which greatly increased the amount of somatic embryos formed. At the same time, an appropriate amount of serine (ser) was added to eliminate the differences in different genotype backgrounds, making the method of the present invention universal for all peanut varieties. A large number of healthy somatic embryos can be obtained in a short period of time, which can be used for genetic improvement breeding of peanuts and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a diagram of the peanut somatic embryo induction pattern;

[0026] Figure 2 Electron microscopic images of embryogenic cell proliferation (a is a comparison of the number of fine sand-like embryogenic cells obtained before and after adding 0.4 g / L gln and 0.2 g / L ser to the embryonic callus induction medium, and b is embryogenic cells induced by 2,4-D and Pic dedifferentiation medium and single 2,4-D dedifferentiation medium);

[0027] Figure 3 This is a diagram of the somatic embryo induction, resistance screening and regeneration process;

[0028] Figure 4 This is a map of the Arahy.3J-Cas9 vector. DETAILED DESCRIPTION

[0029] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in each embodiment of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0030] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0031] Example: A widely applicable and efficient method for inducing peanut somatic embryos

[0032] like Figure 1 As shown, the induction of peanut somatic embryos (the whole process takes about 2-3 months) includes the following steps:

[0033] (1) Pre-cultivation of peanut seeds:

[0034] After disinfection (soaking in 75% alcohol for 1 min), remove the alcohol from peanut seeds (seven varieties: Zhanyou 65, Zhanyou 75, Yueyou 7, Yueyou 13, Guihua 21, Fuhua 9, and Zhonghua 16), wash once with sterile water, add 10% sodium hypochlorite solution and soak for 20 min, rinse with sterile water 5 to 6 times, soak the seeds in sterile water for the last time overnight, take them out and dry them the next day, and place them in MS culture medium for pre-culture for about 5 days.

[0035] (2) Selection of explants:

[0036] Take out the peanut seeds and place them on a cutting plate. Remove the two cotyledons to leave the young embryo. Then, cut the seeds at the junction of the embryo leaflet and the embryo axis (see Figure 1 ) Cut 3-4 thin slices (1-3 mm) from top to bottom with a blade, and place the slices in a dedifferentiation medium (MS+5mg / L 2,4-D+5mg / L pic+0.5mg / L BAP+3% sucrose) for culture. After about 2 weeks of culture, the slices become transparent. At the same time, a single high concentration of 2,4-D (10 mg / L) is used as a control (MS+10mg / L 2,4-D+0.5mg / L BAP+3% sucrose).

[0037] (3) Inducing embryonic callus and promoting the proliferation of embryogenic cells:

[0038] The dedifferentiated transparent sheets were transferred to embryonic callus induction medium [1 / 2MS+5mg / L 2,4D+3mg / LPic+0.2mg / L ZT+ser(0.1g / L, 0.2g / L, 0.3g / L)+gln(0.3g / L, 0.4g / L, 0.5g / L)+2% sucrose] for culture. At the same time, embryonic callus induction medium [1 / 2MS+5mg / L 2,4D+3mg / L Pic+0.2mg / L ZT+2% sucrose] was used as a control. This process was cultured for 2-3 weeks.

[0039] After induction, many fine sand-like embryonic cells can be seen growing out of the transparent sheets ( Figure 2 a left). In contrast, the addition of gln and ser to the embryogenic callus induction medium significantly increased the number of embryogenic cells ( Figure 2 a right). As the culture time increases, these embryogenic cells gradually differentiate and grow into somatic embryos. Figure 2 b It can be seen that compared with the use of high concentration of 2,4-D (10 mg / L) alone, the browning of explants was greatly reduced by the combined use of 2,4-D and Pic for dedifferentiation induction.

[0040] In this process, Agrobacterium infection or gene gun transformation can be carried out. In addition, the concentrations of ser and gln are adjusted according to the peanut oil content and the thickness of the shell. Generally speaking, the thicker the shell, the higher the amount, and vice versa, but too high gln will cause browning.

[0041] (4) Somatic embryo induction and subculture:

[0042] The transparent sand-like embryonic callus was transferred to the somatic embryo induction and subculture medium (1 / 2MS + 3mg / L Pic + 0.2mg / L ZT + 0.1% activated carbon + 2% sucrose) for culture. This process lasted for about 4 weeks until a large number of somatic embryos grew out ( Figure 3 ).

[0043] The transformed vector can then be used for corresponding resistance screening, for example (see Figure 3 ), through the method of Agrobacterium infection (for specific operations, refer to the literature "Yang Yanwen, Optimization of Agrobacterium-mediated Peanut Genetic Transformation System. Master's thesis of Hebei Agricultural University. 2020, p11.; Zhai Qiong, Chen Rongqin, Liang Xiaohua, etc. Establishment and application of a rapid genetic transformation method for peanut. Acta Botanica Sinica, 2022, 57(03)"), the vector carrying Kanna resistance is transformed into the embryo (the transformed vector is Arahy.3J-Cas9, with Kanna screening tag, the vector structure is as follows Figure 4 After 6-8 weeks of Kana resistance screening of the transformed somatic embryos, most of the untransformed somatic embryos gradually turned brown and died, while the resistant positive somatic embryos could continue to be subcultured and proliferated, and finally the positive somatic embryos were germinated and regenerated.

[0044] (5) Somatic embryo germination and regeneration:

[0045] The somatic embryos were transferred to germination medium (1 / 2MS + 0.1% activated carbon + 4mg / L 6BAP + 1% sucrose) and grown under light. After about 2 weeks, the top of the somatic embryos began to turn green and gradually undergo polar growth, that is, buds, true leaves and roots grew respectively. The regenerated seedlings with good growth were transplanted to the soil for growth ( Figure 3 Finally, the somatic embryo induction rate and seedling rate were counted, and the results are shown in Table 1.

[0046] Table 1 Somatic embryo induction of different genotypes of peanut varieties

[0047]

[0048]

[0049] The statistics of the number of somatic embryos in Table 1 are the number of somatic embryos directly induced by each explant after the first proembryonic cell induction. The somatic embryo induction rate is the number of somatic embryos produced divided by the number of explants. However, in actual operation, after the first proembryonic cell proliferation, in order to avoid the center effect, the induced somatic embryos are often cut into small pieces or peeled off before the embryo is subcultured and regenerated. Therefore, the somatic embryos subcultured and proliferated later are often much larger than the number of somatic embryos directly induced, but they are not included in the statistical scope. The seedling rate is the peanut plant directly generated by each explant after somatic embryo induction.

[0050] As can be seen from Table 1, a large number of somatic embryos can be obtained by the method of the present invention, and the somatic embryo induction rate can be as high as 1200.00% (while the induction rate of the current peanut somatic embryo induction method is mainly between 7.5% and 92%), and the technology is stable and applicable to various peanut varieties (including Zhanyou 65, Zhanyou 75, Yueyou 7, Yueyou 13, Guihua 21, Fuhua 9, and Zhonghua 16, etc.). At the same time, the deformed embryos induced by the method of the present invention are very few, and basically all of them can develop into seedlings, which has great application prospects in the genetic improvement of peanuts.

[0051] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions and variations of these embodiments are made without departing from the principles and spirit of the present invention, and still fall within the protection scope of the present invention.

Claims

1. A widely applicable and efficient peanut somatic embryo induction and transformation method, characterized in that: The following steps are involved: S1. After pre-culturing peanut seeds, two cotyledons were removed to leave young embryos, 3-4 thin slices were cut from top to bottom at the junction of the embryo leaflet and the embryo axis, and the cut slices were cultured in a dedifferentiation medium until the slices became transparent. The dedifferentiation medium included MS, 5 mg / L 2,4-D, 5 mg / L Pic, 0.5 mg / L 6-BA and 3% sucrose; S2, converting the dedifferentiated transparent thin slices to an embryonic callus induction medium, and culturing until the transparent thin slices grow many sand-like embryogenic cells, wherein the embryonic callus induction medium includes 1 / 2MS, 5 mg / L 2,4-D, 3 mg / L Pic, 0.2 mg / L ZT, 0.05-0.1 g / L ser, 0.3-0.5 g / L gln and 2% sucrose; S3, transferring the transparent sand-like embryonic callus to a somatic embryo induction and subculture medium for culture until a large number of somatic embryos grow out, wherein the somatic embryo induction and subculture medium comprises 1 / 2MS, 3 mg / L Pic, 0.2 mg / L ZT, 0.1% activated carbon and 2% sucrose; S4. Transfer the somatic embryos to a germination medium and grow them under light. When the tops of the somatic embryos begin to turn green, replace them with a growth medium for seedling culture. The germination medium includes 1 / 2MS, 0.1% activated carbon and 1% sucrose. The growth medium includes 1 / 2MS, 0.1% activated carbon, 4 mg / L 6-BA and 1% sucrose.

2. A widely applicable and efficient peanut somatic embryo induction and transformation method according to claim 1, characterized in that: The varieties of peanuts include Zhanyou 65, Zhanyou 75, Yueyou 7, Yueyou 13, Guihua 21, Fuhua 9 and Zhonghua 16.

3. A widely applicable and efficient peanut somatic embryo induction and transformation method according to claim 1, characterized in that: The thickness of the sheet in S1 is 1-3 mm.

4. A widely applicable and efficient peanut somatic embryo induction and transformation method according to claim 1, characterized in that: S1 The peanut seed pre-culture is to culture the peanut seeds in MS medium for 4-6 days.

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