Method for inducing mongolian mongolian mongolian stem cells to form regenerated plants

Abstract

The invention belongs to the field of plant stem cell regeneration, and discloses a method for inducing tetrasynthum mongolicum stem cells to form regenerated plants.The method comprises the steps that tetrasynthum mongolicum stem cells are obtained after tetrasynthum mongolicum explants are cultured through an induction culture medium and a subculture medium, and after the tetrasynthum mongolicum stem cells are subjected to suspension culture, callus tissue is obtained through induction of a callus culture medium; inoculating the calluses into a culture medium containing a regeneration factor REF1, differentiating into embryogenic calluses of the tetrasyna mongolica, and transferring the embryogenic calluses of the tetrasyna mongolica into an embryoid induction culture medium to obtain embryoids of the tetrasyna mongolica; inoculating the embryoid into a bud proliferation culture medium to obtain clumpy buds; and inoculating the clumpy buds into an elongation rooting culture medium for subculture to obtain the tetracalamus mongolicus seedlings. Compared with a traditional method for performing tissue culture by using sterile seedlings, the method disclosed by the invention has the advantages that stem cells are utilized for performing regeneration on the tetrasyna mongolica plant for the first time, excellent cell division and differentiation capabilities are shown, the characters of a female parent can be kept, and long-term growth can be maintained.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant tissue culture and plant regeneration, and particularly relates to a method for inducing the formation of Tetraena mongolica stem cells into regenerated plants. Background Art

[0002] Plant biotechnology involves applying the principles and technologies in the fields of biology, agronomy, biochemistry, molecular biology, etc. to conduct research and applications on plant genetic improvement, reproduction control, new variety cultivation, pest control, etc. The plant tissue culture technology has gradually become mature. Nowadays, plant biotechnology pays more attention to ecological environmental protection and sustainable development, and it has been widely applied in many fields such as agriculture, forestry, horticulture, medicine, food, etc., such as the large-scale propagation of precious flowers and the protection of rare and endangered plants through tissue culture technology. Tetraena mongolica is mainly distributed in Hangjin Banner and Etuoke Banner of Ordos, as well as Wuhai City, the eastern part of Alxa Left Banner, the northern part of Helan Mountain and Shizuishan City. Due to its rarity and uniqueness, it occupies a unique and crucial position in the ecosystem. It is not only crucial for maintaining ecological balance, but also plays a significant role in wind prevention and sand fixation, and improving soil structure by fixing sand, reducing wind erosion and water and soil loss. The main reasons for the endangerment of the Tetraena mongolica population are weak reproductive ability. The geographical factors of drought and salinity often inhibit the development and germination of seeds, and only 1% of the seeds can mature and reproduce, resulting in a weakened sexual reproduction ability; Tetraena mongolica is vulnerable to pests such as Apriona swainsoni and rats; the branches of Tetraena mongolica are rich in oil and are called "oil firewood", and because they are flammable, they have been used as firewood for a long time, resulting in a large amount of deforestation and damage, so that the population number of Tetraena mongolica has decreased by about 70%, and its habitat area has also shrunk by more than 30%. The severity of habitat degradation is worrying. Existing technologies also have methods of inducing plant regeneration by cutting technology and callus. However, during the cutting process, the survival rate of Tetraena mongolica is restricted by culture conditions and is generally low, making it difficult to efficiently obtain a large number of seedlings. Although directly using the callus of Tetraena mongolica can induce plant regeneration, its stability is poor, it is difficult to stably maintain the integrity of genetic information and parental traits for a long time, and the cell system is difficult to maintain for a long time, and the recovery rate after cryopreservation is also low. These factors limit its application in large-scale obtaining of Tetraena mongolica seedlings to repair the desertified ecological environment. Therefore, a new method for regenerating Tetraena mongolica plants is needed. Summary of the Invention

[0003] To solve the above problems, the present invention provides a method for inducing Tetraena mongolica stem cells to form regenerated plants. By using Tetraena mongolica stem cells as the key resource, callus is obtained through the induced culture of Tetraena mongolica stem cells. Under the action of the regeneration factor REF1, the induction rate of embryogenic callus and embryoids is effectively increased, and regenerated plants are successfully cultivated, realizing the rapid propagation of Tetraena mongolica.

[0004] To achieve the above object, the technical solution of the present invention is as follows.

[0005] A method for inducing Tetraena mongolica stem cells to form regenerated plants, comprising the following steps:

[0006] Induce and culture the Tetraena mongolica explant in the first induction medium to obtain cambium stem cells; transfer the cambium stem cells to the subculture medium for subculture to obtain Tetraena mongolica stem cells with stable growth. After suspension culture of the Tetraena mongolica stem cells, callus is obtained through induction in the callus-containing medium;

[0007] Inoculate the callus into the second induction medium containing the regeneration factor REF1 for culture, and promote the gene conversion rate of embryogenic callus through the regeneration factor REF1 to differentiate into embryogenic callus;

[0008] Transfer the embryogenic callus to the third induction medium containing the regeneration factor REF1 for culture, promote the differentiation of embryoids through the regeneration factor REF1 to obtain embryoids of Tetraena mongolica. Inoculate the embryoids into the bud proliferation medium to obtain Tetraena mongolica cluster buds, and subculture the Tetraena mongolica cluster buds in the elongation and rooting medium to obtain Tetraena mongolica seedlings, that is, Tetraena mongolica regenerated plants;

[0009] In the embryogenic callus induction medium and the tissue induction medium, the concentration of the regeneration factor REF1 is 100 nM to 200 nM.

[0010] The present invention obtains callus through the induced culture of Tetraena mongolica stem cells. The obtained callus is cultured in the second induction medium containing 100 nM to 200 nM of the regeneration factor REF1 to improve the gene conversion rate of the callus, greatly improve the regeneration ability of the callus, and then promote the differentiation of embryos through culture in the third induction medium containing 100 nM to 200 nM of the regeneration factor REF1, improve the differentiation speed and success rate of embryoids, reduce the probability of embryoids being deformed during development, and thus effectively improve the regeneration ability of the callus, realizing the rapid propagation of Tetraena mongolica.

[0011] In another preferred embodiment, the induction medium is an MS medium containing 30 g / L to 40 g / L sucrose, 500 mg / L to 700 mg / L hydrolyzed lactalbumin, 3 g / L to 4 g / L gellan gum, 0.5 mg / L to 1 mg / L 6-benzylaminopurine, and 0.25 mg / L to 1 mg / L 2,4-dichlorophenoxyacetic acid; the pH value of the induction medium is 5.75 to 5.85;

[0012] The conditions for the induction culture are: temperature 25°C to 30°C, humidity 60% to 80%, and light culture for 8 h / d to 12 h / d.

[0013] In another preferred embodiment, the subculture medium is an MS medium containing 30 g / L to 40 g / L sucrose, 500 mg / L to 700 mg / L hydrolyzed lactalbumin, 3 g / L to 4 g / L gellan gum, 0.1 mg / L to 1 mg / L 6-benzylaminopurine, and 0.1 mg / L to 2 mg / L 2,4-dichlorophenoxyacetic acid, and the pH value of the induction medium is 5.75 to 5.85;

[0014] The culture conditions for the subculture are: temperature 25°C to 30°C, humidity 60% to 80%, and light culture for 8 h / d to 12 h / d.

[0015] In another preferred embodiment, the specific process of the suspension culture is as follows:

[0016] 50 g to 100 g of Tetraena mongolica Maxim stem cells are inoculated into each liter of the suspension medium and continuously cultured under light for 15 d to 20 d;

[0017] The suspension medium is an MS medium containing 10 g / L to 30 g / L sucrose, 500 mg / L to 700 mg / L hydrolyzed lactalbumin, 3 g / L to 4 g / L gellan gum, 0.1 mg / L to 3 mg / L 6-benzylaminopurine, and 0.25 mg / L to 1 mg / L 2,4-dichlorophenoxyacetic acid, and the pH value of the suspension medium is 5.75 to 5.85;

[0018] The conditions for the suspension culture are: temperature 25°C to 30°C, humidity 60% to 80%, and continuous light for 15 d to 20 d.

[0019] In another preferred embodiment, the callus medium is an MS medium containing 10 g / L to 30 g / L sucrose, 500 mg / L to 600 mg / L hydrolyzed lactalbumin, 3 g / L to 4 g / L gellan gum, 0.8 mg / L to 1 mg / L 6-benzylaminopurine, 0.5 mg / L to 1 mg / L 2,4-dichlorophenoxyacetic acid, and the pH value of the callus medium is 5.75 to 5.85;

[0020] The culture conditions of the callus medium are as follows: temperature 25°C - 30°C, humidity 60% - 80%, and light culture for 10 h / d - 12 h / d.

[0021] In another preferred embodiment, the second induction medium is an MS medium containing 30 g / L - 40 g / L sucrose, 500 mg / L - 700 mg / L hydrolyzed milk protein, and 3 g / L - 4 g / L gellan gum, and the pH value of the second induction medium is 5.75 - 5.85;

[0022] The culture conditions of the second induction medium are as follows: temperature 25°C - 30°C, humidity 60% - 80%, and light culture for 10 h / d - 12 h / d.

[0023] In another preferred embodiment, the third induction medium is an MS medium containing 30 g / L - 40 g / L sucrose, 500 mg / L - 700 mg / L hydrolyzed milk protein, 3 g / L - 4 g / L gellan gum, 0.1 mg / L - 0.5 mg / L 6-benzylaminopurine, and 0.25 mg / L - 0.3 mg / L 2,4-dichlorophenoxyacetic acid, and the pH value of the third induction medium is 5.75 - 5.85;

[0024] The culture conditions of the third induction medium are as follows: temperature 25°C - 30°C, humidity 60% - 80%, and light culture for 10 h / d - 12 h / d.

[0025] In another preferred embodiment, the shoot proliferation medium is a B5 medium containing 30 g / L - 40 g / L sucrose, 500 mg / L - 700 mg / L hydrolyzed milk protein, 3.6 g / L - 4.0 g / L gellan gum, and 0.1 mg / L - 1 mg / L naphthaleneacetic acid, and the pH value of the shoot proliferation medium is 5.75 - 5.85;

[0026] The culture conditions of the shoot proliferation medium are as follows: temperature 25°C - 30°C, humidity 60% - 80%, and light culture for 8 h / d - 12 h / d.

[0027] In another preferred embodiment, the rooting medium is a B5 medium containing 30 g / L - 40 g / L sucrose, 500 mg / L - 700 mg / L hydrolyzed milk protein, 3.6 g / L - 4 g / L gellan gum, 0.1 mg / L - 1 mg / L 6-benzylaminopurine, 1 mg / L - 2 mg / L naphthaleneacetic acid, and 1 g / L - 1.5 g / L activated carbon, and the pH value of the rooting medium is 5.75 - 5.85;

[0028] The culture conditions of the rooting medium are as follows: temperature 25°C - 30°C, humidity 60% - 80%, and light culture for 10 h / d - 12 h / d.

[0029] In another preferred embodiment, the specific process for obtaining the Tetraena mongolica explants is as follows:

[0030] Cut the Tetraena mongolica branches into segments of 5 cm to 8 cm. After sterilization, cut them into slices with a thickness of 2 mm to 4 mm to obtain the Tetraena mongolica explants;

[0031] The specific process of the sterilization is as follows: rinse with water for 0.5 h to 2 h, then soak successively in 10 wt% hydrogen peroxide for 1 min to 2 min, 75 wt% ethanol solution for 1 min to 2 min, rinse with sterile water 2 to 3 times, soak in 0.05 wt% mercuric chloride solution for 10 min to 20 min, soak in sterile water for 1 min to 3 min, and then rinse with sterile water 5 to 6 times.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The present invention obtains the stem cells of Tetraena mongolica from the branches of Tetraena mongolica, and then induces the formation of callus, and gradually develops into embryogenic callus, embryoids, and cluster buds, and finally realizes the whole process of regenerating plants. By adding 100 nM to 200 nM of the regeneration factor REF1 in the second induction medium and the third induction medium, the induction rate of embryogenic callus and embryoids is increased, and the regeneration ability of callus is greatly improved, thus effectively avoiding the problems of poor stability and difficulty in maintaining the cell system for a long time in the process of inducing plants by using callus in the prior art, providing a solid foundation for constructing an efficient and genetically stable somatic embryo regeneration system, and having far-reaching significance for the germplasm resource protection, genetic stability maintenance and biotechnological tool development of Tetraena mongolica. The method in the present invention has the characteristics of synchronous growth, stable genetic information, stable biosynthesis of secondary metabolites and high yield, and well overcomes the defect that the dedifferentiated cell culture system is prone to genetic variation and causes changes in its physiological and biochemical characteristics.

[0034] The present invention has established an efficient induction and long-term stable in vitro culture method for plant stem cells, callus and adventitious roots derived from Tetraena mongolica leaf explants. The plant stem cells, callus and adventitious root systems are all suitable for suspension culture. The stem cells exist as dispersed single cells or small cell clusters; the callus grows as large cell masses; the adventitious roots grow while maintaining the root organ morphology. The experimental results show that the growth rates of Tetraena mongolica stem cells and callus are 1080% and 815% respectively. The growth rate of Tetraena mongolica stem cells is the best, followed by the callus. Therefore, Tetraena mongolica stem cells are more suitable for large-scale culture in a bioreactor. The present invention can effectively reduce contamination by sterilizing the Tetraena mongolica leaf explants multiple times, and significantly improves the induction success rate. Description of the Drawings

[0035] Figure 1 It is a diagram of the induction process of Tetraena mongolica stem cells; among them, A is a picture of a Tetraena mongolica branch; B is a picture of a Tetraena mongolica explant; C is a picture of Tetraena mongolica cultured in an induction medium for 5 days; D is a picture of Tetraena mongolica cultured in an induction medium for 12 days; E is a picture of Tetraena mongolica callus; F is a picture of Tetraena mongolica stem cells.

[0036] Figure 2 It is a microscopic observation diagram of Tetraena mongolica stem cells and callus; among them, A is a diagram of Tetraena mongolica stem cells magnified 20 times; B is a diagram of a single Tetraena mongolica stem cell magnified 40 times; C is a diagram of Tetraena mongolica callus magnified 10 times; D is a diagram of a single cell of Tetraena mongolica callus magnified 40 times.

[0037] Figure 3 It is a growth curve diagram of Tetraena mongolica stem cells and callus. In the figure, CMCs represents Tetraena mongolica stem cells, and DDCs represents callus. The descriptions in the following figures are the same as this.

[0038] Figure 4 It is a diagram of the relationship between the cell survival rate of Tetraena mongolica stem cells and callus after treatment with different concentrations of bleomycin; among them, A is a diagram of the relationship between the cell survival rate of Tetraena mongolica stem cells and callus after treatment with 150 μg / mL bleomycin; B is a diagram of the relationship between the cell survival rate of Tetraena mongolica stem cells and callus after treatment with 300 μg / mL bleomycin.

[0039] Figure 5 It is a diagram of the comparison result of the cell survival rate of Tetraena mongolica stem cells and callus after cryopreservation and resuscitation.

[0040] Figure 6 It is a diagram of Tetraena mongolica embryogenic callus; among them, A is a picture of Tetraena mongolica embryogenic callus; B is a microscopic observation diagram of Tetraena mongolica embryogenic callus magnified 10 times.

[0041] Figure 7 It is a diagram of Tetraena mongolica embryoid.

[0042] Figure 8 It is a diagram of Tetraena mongolica cluster buds.

[0043] Figure 9 It is a diagram of Tetraena mongolica seedlings. Detailed implementation mode

[0044] Next, this specification will elaborate on the technical solutions of the present invention in detail and completely based on the data provided in the embodiments of the present invention. Obviously, the embodiments described here only represent a part of the numerous embodiments of the present invention, not all of them. All other embodiments that can be thought of by those of ordinary skill in the art without creative efforts based on these embodiments of the present invention should be regarded as falling within the scope of the present invention.

[0045] It should be clear that the professional terms involved in this article are only for the purpose of elaborating specific embodiments, and are not intended to limit the protection scope of the present invention. Unless otherwise specified, various raw materials, reagents, instruments and equipment mentioned in the subsequent embodiments of the present invention can be purchased on the market or prepared by existing technical methods.

[0046] In 2024, the research group of Li Chuanyou from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, for the first time identified the primary wound signal molecule that induces plant regeneration: regeneration factor REF1 (REGENERATION FACTOR1), systematically revealed the signal transduction network of REF1 regulating tissue repair and organ regeneration, and at the same time proved the great application value of REF1 in the fields of plant transgenesis, plant germplasm resource preservation, and gene editing. In the process of callus culture of the present invention, adding the regeneration factor REF1 can improve the gene conversion rate of callus, greatly improve the regeneration ability of callus; adding the regeneration factor REF1 in the process of embryoid culture can promote the differentiation of embryos, improve the differentiation speed and success rate of embryoids, and reduce the probability of embryoids being deformed during development. Thus, it greatly improves the regeneration ability of callus, providing a solid foundation for constructing an efficient and genetically stable somatic embryo regeneration system. The method in the present invention has the characteristics of growth synchronization, stable genetic information, stable biosynthesis of secondary metabolites and high yield, and well overcomes the defect that the dedifferentiated cell culture system is prone to genetic variation and causes changes in its physiological and biochemical characteristics.

[0047] In the following examples, the regeneration factor REF1 was synthesized by Sangon Biotech (Shanghai) Co., Ltd., with a purity > 90%.

[0048] 6-Benzylaminopurine is denoted as 6-BA, naphthaleneacetic acid is denoted as NAA, and 2,4-dichlorophenoxyacetic acid is denoted as 2,4-D.

[0049] Example 1

[0050] A method for inducing the formation of regenerated plants from Tetraena mongolica stem cells specifically includes the following steps:

[0051] S1. Explant sterilization: In the early morning of March to May in spring, pick fresh Tetraena mongolica, a plant of the family Zygophyllaceae and genus Tetraena, which is 5 to 8 months old. Take annual branches and longitudinally cut them into small sections of 6 cm, as Figure 1As shown in A in [reference], after rinsing with tap water for 0.5 h, the surface moisture was then sucked off; the small segments were placed in a sterile laminar flow bench and successively soaked in 10 wt% hydrogen peroxide for 1 min, 75 wt% ethanol solution for 1 min, rinsed twice with sterilized distilled water, soaked in 0.05 wt% mercuric chloride solution for 15 min, then soaked in sterilized distilled water for 3 min, and rinsed 6 times with sterilized distilled water. Then they were put into a 150 mg / L citric acid solution and soaked for 1 min. After taking them out, they were transversely cut to remove the pith, and finally two slices with a thickness of 4 mm containing xylem with vascular bundles, phloem, and cambium were obtained, namely the Tetraena mongolica explants, as Figure 1 shown in B in [reference].

[0052] S2. Isolation and differentiation culture of Tetraena mongolica stem cells and callus: The cross-section of the Tetraena mongolica explant was closely attached to the surface of the first induction medium for cambium stem cell induction culture for 8 days; the induction culture conditions were: temperature 25 °C, humidity 75% - 80%, the light source was a full-spectrum supplementary light lamp, the light intensity was 2883.8 lx, and the light time was 12 h / d.

[0053] The first induction medium was based on the MS medium, supplemented with 30 g / L sucrose, 4 g / L gellan gum, 500 mg / L hydrolyzed milk protein, 1 mg / L 6-BA, and 0.5 mg / L 2,4-D. The pH value of the first induction medium was 5.75 - 5.85; the formula of the MS medium is shown in Table 1.

[0054] Table 1 MS medium formula

[0055] Component Concentration <![CDATA[KNO 3 > 2500 mg / L <![CDATA[(NH 4 ) 2 NO 3 > 134 mg / L <![CDATA[KH 2 PO 4 ·5H 2 O]]> 150 mg / L <![CDATA[MgSO 4 ·7H 2 O]]> 250 mg / L <![CDATA[CaCl 2 > 113.2 mg / L <![CDATA[C 6 H 12 O 6 > 100 mg / L KI 0.75 mg / L <![CDATA[Sodium 2 Molybdenum Oxide 4 Phosphate Oxide 4 ·2 Water 2 O]]> 0.25 mg / L <![CDATA[CuSO 4 ·5H 2 O]]> 0.025 mg / L <![CDATA[CoCl 2 ·6H 2 O]]> 0.025 mg / L <![CDATA[Na-EDTA·2H 2 O]]> 37.3 mg / L <![CDATA[FeSO 4 ·7H 2 O]]> 27.8 mg / L <![CDATA[C 12 H 17 ClNOS·HCl]]> 10.0 mg / L <![CDATA[C 8 H 12 ClNO 3 > 1.0 mg / L

[0056] When preparing the MS liquid medium, according to the formula in Table 1, distilled water was used to dissolve and mix them respectively and then made up to 1 L, the pH was adjusted to 5.75, and it was autoclaved at 121 °C for 20 min and then cooled to obtain the MS liquid medium; 4.0 g / L gellan gum was added to the MS liquid medium, sterilized, and cooled to obtain the MS solid medium, and all the following MS medium formulas were like this. And the induction medium, isolation medium, callus medium, embryogenic callus induction medium, and embryo-like body induction medium are all solid media.

[0057] After the induction culture, Tetraena mongolica cambium stem cells were obtained, and the specific results are shown in Table 2.

[0058] Table 2 Results of the induction success rate of Tetraena mongolica branches

[0059]

[0060] As can be seen from Table 2, the induction success rate of Tetraena mongolica branches under this sterilization condition and hormone level is between 87.5% and 92.5%, with a relatively high success rate, which is suitable as the induction condition for in vitro culture of Tetraena mongolica. As Figure 1 shown in C, D, and F in Figure 1 , in the induction medium, the cambial stem cells in the root tuber rapidly divide and grow, and the cells continuously proliferate to form a soft and loose cell mass in appearance. The cell mass and the explant residues are gradually peeled off naturally to obtain cells for in vitro culture, showing a light yellowish-white color. In the induction medium, the cambial stem cells and the surrounding cells rapidly divide and differentiate. As the cells proliferate, light green granular cell masses can be observed, which are dedifferentiated cells, namely callus, as

[0061] shown in E in Figure 2 .

[0062] The cambial stem cell mass and tissue are transferred to the subculture medium for subculture, and the subculture medium is replaced every 14 days. This is repeated for continuous culture for 6 months until stable-growing Tetraena mongolica stem cells and callus are obtained. Under the microscope, the cell characteristics are that the cells have small and numerous vacuoles, and the cells are single cells, dispersed and not in clumps, as

[0063] shown in A and B in

[0064] The subculture medium is based on the MS medium, supplemented with 30 g / L sucrose, 4.0 g / L gellan gum, 500 mg / L lactalbumin hydrolyzate, 0.1 mg / L 6-BA, and 0.1 mg / L 2,4-D. The pH value of the subculture medium is 5.8. The conditions for subculture are: temperature 25°C, humidity 60%, light intensity 2883.8 lx, and light time 12 h / d.

[0065] The callus suspension medium is based on MS medium, supplemented with 30 g / L sucrose, 4 g / L gellan gum, 500 mg / L hydrolyzed milk protein, 1 mg / L 6-BA and 0.5 mg / L 2,4-D. The pH value of the callus suspension medium is 5.85. The culture conditions are as follows: the cell inoculation amount is 60 g / L, cultured in a 250 mL shaking flask, at a temperature of 25 °C, a humidity of 60%, a light intensity of 2883.8 lx, continuous light for 20 d, and a rotation speed of 120 r / min.

[0066] Growth curve Figure 3 As shown, the Tetraena mongolica stem cells and callus reached the peak of growth at 12 d. After measurement, the growth rate of Tetraena mongolica stem cells was 1080% and that of the callus was 815%. It shows that under the same culture conditions, the growth rate of stem cells is significantly higher than that of the callus. To further determine the activity of stem cells and callus, the following experiments were conducted, as follows.

[0067] Bleomycin sensitivity experiment: The Tetraena mongolica stem cells and callus were suspension-cultured for 12 d. Take 2 mL of the cell suspension and evenly place it in 12-well plates respectively. Add 150 μg / mL and 300 μg / mL bleomycin solutions to the two groups of 12-well plates respectively. After shaking culture for 24 h and 48 h, add 12 μg / mL fluorescein diacetate, shake and mix well, observe with a fluorescence microscope, and calculate the cell mortality rate. The calculation formula for the cell mortality rate is: Cell mortality rate (%) = (total number of cells - number of live cells) / total number of cells * 100%.

[0068] The results are as Figure 4 shown. After treatment with bleomycin, the mortality rate of Tetraena mongolica stem cells is significantly higher than that of the callus, and it is more sensitive to bleomycin, showing the typical characteristics of plant stem cells.

[0069] Cryopreservation and recovery experiment: Use the Tetraena mongolica stem cells and callus cultured for 12 d, put them into the cryoprotectant for cryopreservation. The formula of the cryoprotectant is MS medium containing 0.5 M glycerol, 0.5 M dimethyl sulfoxide, 30 g / L sucrose, 500 mg / L hydrolyzed milk protein, 1 mg / L 6-BA, and 0.50 mg / L 2,4-D; the cell inoculation amount is 6 g / 100 mL. After maintaining the suspension cells treated with the cryoprotectant at -4 °C for 30 minutes, put them into a -20 °C refrigerator for 3 h, and then immerse them in liquid nitrogen for freezing. After 30 min of liquid nitrogen treatment, take out the cells and thaw the cells in a 40 °C constant temperature water bath under sterile conditions for 2 min. For cell regrowth, a sterile funnel and filter paper were used for the cell suspension. The filtered cells were placed on a solid growth medium including filter paper and stabilized at room temperature for 30 min, and then transferred to a fresh subculture medium again. The survival rate of Tetraena mongolica stem cells after cryopreservation and recovery is much better than that of the callus, asFigure 5 As shown, it indicates that the stem cell activity of Tetraena mongolica is much higher than that of callus. Therefore, Tetraena mongolica stem cells are selected for regenerated plants as follows.

[0070] S3. Callus induction: Select Tetraena mongolica stem cells that have been suspension-cultured for 14 days and inoculate them into the callus medium. The amount of cells inoculated in each bottle is 3 g. Transfer them to the induction medium every 14 days for continuous culture, and replace the induction medium every 14 days. Repeat this continuous culture for 3 cycles of 14 days to obtain Tetraena mongolica callus with stable growth. Count the induction rate and differentiation effect of Tetraena mongolica callus for observation and comparison. In the callus medium, stem cells and their adjacent cells rapidly divide and differentiate. As the number of cells increases, light green granular cell clusters can be observed under the microscope. These cells are characterized by containing a large vacuole, which indicates that they are dedifferentiated cells, namely callus, as shown in Figure 2 C and D in

[0071] The said callus medium is based on MS medium, supplemented with 30 g / L sucrose, 4 g / L gellan gum, 500 mg / L hydrolyzed milk protein, 1 mg / L 6-BA, 0.5 mg / L 2,4-D, and the pH value of the callus medium is 5.85.

[0072] The culture conditions are: temperature 26 °C, humidity 70%, light intensity 2883.8 lx, and light time 12 h / d.

[0073] S4. Embryogenic callus induction: Take Tetraena mongolica callus and inoculate it into the second induction medium. The amount of cells inoculated in each bottle is 3 g; replace the induction medium every 14 days. Repeat this continuous culture for 3 cycles of 14 days to obtain embryogenic callus. Count the induction rate and differentiation effect of embryogenic callus, as shown in Table 3.

[0074] The said second induction medium is based on MS medium, supplemented with 30 g / L sucrose, 4 g / L gellan gum, 500 mg / L hydrolyzed milk protein, and 100 nM regeneration factor REF1. The pH value of the second induction medium is 5.75 - 5.85. The culture conditions are: temperature 26 °C, humidity 70%, light intensity 2883.8 lx, and light time 12 h / d.

[0075] Table 3 Results of embryogenic callus induction rate

[0076]

[0077] As shown in Table 3, the average induction rate of embryogenic callus was 72%. In the callus induction medium, the callus and the surrounding cells divided and differentiated rapidly. With cell proliferation, it was observed that the light green cells became hard and formed large masses, and then turned dark green. Under the microscope, the cells became dense and showed tissue-like differentiation, which was embryogenic callus, as Figure 6 shown.

[0078] S5. Embryoid induction: Select the embryogenic callus of Tetraena mongolica cultured for 14 days and inoculate it into the third induction medium for culture. The amount of inoculated cells in each bottle is 3 g; replace the induction medium every 14 days, and repeat the continuous culture for 3 periods of 14 days to obtain embryoids with stable growth. The embryoid induction rate and differentiation effect are shown in Table 4;

[0079] The third induction medium is based on MS medium, supplemented with 30 g / L sucrose, 4 g / L gellan gum, 500 mg / L hydrolyzed milk protein, 0.1 mg / L 6-BA, 0.25 mg / L 2,4-D, and 100 nM regeneration factor REF1. The pH value of the third induction medium is 5.85. The culture conditions are: temperature 26°C, humidity 70%, light intensity 2883.8 lx, and light time 12 h / d.

[0080] Table 4 Results of embryoid induction rate

[0081]

[0082] As shown in Table 4, the average induction rate of embryoids under this culture condition was 73%. In the embryoid induction medium, the embryogenic callus and the surrounding cells divided and differentiated rapidly. With cell proliferation, it was observed that the large masses of light green cells became soft again and turned yellowish green. The embryogenic callus differentiated into individual somatic embryos. Under the microscope, the embryogenic callus had the rudiment of organs, which was the embryoid, as Figure 7 shown.

[0083] S6. Shoot cluster induction: Select the embryoids cultured for 14 days, transfer the whole piece of callus with embryoids to the shoot proliferation medium for continuous culture to differentiate adventitious shoots. Replace the shoot proliferation medium every 14 days for the adventitious shoots differentiated. After repeating 3 times, multiple shoot clusters are formed; transfer the multiple shoot clusters to the induction medium for continuous culture, replace the induction medium every 14 days, and repeat the continuous culture for 3 periods of 14 days to obtain shoot clusters with stable growth. The adventitious shoot differentiation rate is shown in Table 6.

[0084] The culture conditions are: temperature 23°C, humidity 70%, light intensity 2883.8 lx, and light time 12 h / d,

[0085] The shoot proliferation medium is based on the B5 medium, supplemented with 40 g / L sucrose, 4 g / L gellan gum, 500 mg / L hydrolyzed milk protein, and 0.10 mg / L NAA. The pH value of the shoot proliferation medium is 5.85.

[0086] The solvent of the B5 medium is water, and the formula is shown in Table 5.

[0087] Table 5 Formula of B5 Medium

[0088] Component Concentration <![CDATA[KNO 3 > 2500 mg / L <![CDATA[(NH 4 ) 2 NO 3 > 134 mg / L <![CDATA[KH 2 PO 4 ·5H 2 O]]> 150 mg / L <![CDATA[MgSO 4 ·7H 2 O]]> 500 mg / L <![CDATA[CaCl 2 ·2H 2 O]]> 150 mg / L <![CDATA[ZnSO 4 ·7H 2 O]]> 2.0 mg / L KI 0.75 mg / L <![CDATA[Sodium 2 Molybdenum Oxide 4 Phosphate Oxide 4 ·2 Water Molecules 2 O]]> 0.25 mg / L <![CDATA[CuSO 4 ·5H 2 O]]> 0.025 mg / L <![CDATA[CoCl 2 ·6H 2 O]]> 0.025 mg / L <![CDATA[FeNa 2 -EDTA]]> 28.0 mg / L <![CDATA[H 2 BO 2 > 3.0 mg / L <![CDATA[MnSO 4 ·4H 2 O]]> 10.0 mg / L

[0089] Table 6 Results of Adventitious Bud Differentiation Rate

[0090]

[0091] As shown in Table 6, the average adventitious bud differentiation rate of Tetraena mongolica embryoids under this culture condition is 76%. In the shoot proliferation culture, the embryoids divide and differentiate rapidly. As the embryoids germinate, it can be observed that the embryoids continuously develop into torpedo embryos, heart-shaped embryos, and bud protrusions, as Figure 8 shown.

[0092] S7. Induction of Tetraena mongolica seedlings: When the cluster buds grow to 1.5 - 2 cm, 2 - 3 small buds together with the callus are cut from the bud cluster to obtain small seedling clusters. The small seedling clusters are inoculated into the rooting medium, with 3 clusters in each bottle. The rooting medium is replaced every 14 days, and this is repeated continuously for 3 cycles of 14 days to obtain Tetraena mongolica seedlings with stable growth. The success rate of test-tube seedlings is counted, and the results are shown in Table 7.

[0093] The rooting medium is based on the MS medium, supplemented with 40 g / L sucrose, 4 g / L gellan gum, 500 mg / L hydrolyzed milk protein, 1 mg / L NAA, 0.1 mg / L 6 - BA, and 1.5 g / L activated carbon. The pH value of the rooting medium is 5.85. The culture conditions are: temperature 23°C, humidity 70%, light intensity 5767.6 lx, and light time 12 h / d.

[0094] Table 7 Results of Rooting Rate of Test-Tube Seedlings

[0095]

[0096] As can be seen from Table 7, the average rooting rate of test-tube seedlings is 93%. Under this culture condition, almost all the Tetraena mongolica buds take root to form small seedlings, as Figure 9 shown.

[0097] Example 2

[0098] A method for inducing the formation of regenerated plants from Tetraena mongolica stem cells, except that the plant hormones in the induction medium are 0.50 mg / L 6-BA and 0.25 mg / L 2,4-D, which are different from those in Example 1, and the remaining steps are the same as those in Example 1. Among them, the results of the cell induction success rate are shown in Table 8.

[0099] Table 8 Cell induction success rate

[0100]

[0101]

[0102] As can be seen from Table 8, the induction success rate of Tetraena mongolica branches under this sterilization condition and hormone level is on average 93%, with a high success rate, which is suitable as the induction condition for in vitro culture of Tetraena mongolica. In the induction medium, the cambium stem cells in the tuberous roots divide and grow rapidly, and the cells continuously proliferate to form a soft and loose cell mass. The cell mass and the explant residues are gradually peeled off naturally to obtain in vitro cultured cells, which are light yellowish-white. The results of the embryogenic callus induction rate are shown in Table 9.

[0103] Table 9 Embryogenic callus induction rate

[0104]

[0105] As shown in Table 9, the average embryogenic callus induction rate is 82%. In the callus induction medium, the callus and its surrounding cells divide and differentiate rapidly. With the proliferation of cells, it can be observed that the light green cells form large masses and become hard, and then turn dark green. Under the microscope, the cells become dense and show tissue-like differentiation, which is the embryogenic callus. The results of the embryoid induction rate are shown in Table 10.

[0106] Table 10 Embryoid induction rate

[0107]

[0108] As shown in Table 10, the average embryoid induction rate under this culture condition is 91%. In the embryoid induction medium, the embryogenic callus and its surrounding cells divide and differentiate rapidly. With the proliferation of cells, it can be observed that the large masses of light green cells become soft again and turn yellowish-green. The embryogenic callus differentiates into individual somatic embryos. Under the microscope, the embryogenic callus has organ primordia, which is the embryoid. The results of the adventitious bud differentiation rate are shown in Table 11.

[0109] Table 11 Adventitious bud differentiation rate

[0110]

[0111] The rooting rate of test-tube seedlings is shown in Table 12.

[0112] Table 12 Rooting rate of test-tube plantlets

[0113]

[0114] As can be seen from Table 12, the average rooting rate of test-tube plantlets is 87%, and under this culture condition, almost all the buds of Tetraena mongolica have rooted to form small seedlings.

[0115] As can be seen from the above practice, for the induction medium, when the concentration of 6-BA is 0.50 mg / L and the concentration of 2,4-D is 0.25 mg / L, the success rate of cell induction, the induction rate of embryogenic callus, the induction rate of embryoids, and the differentiation rate of adventitious buds are all higher than those when the concentration of 6-BA is 1 mg / L and the concentration of 2,4-D is 0.5 mg / L. However, its rooting rate is lower than that when the concentration of 6-BA is 1 mg / L and the concentration of 2,4-D is 0.5 mg / L.

[0116] Obviously, those skilled in the art can make various modifications and variations to the present invention without departing from the spirit and scope of the present invention. If these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also incorporates these modifications and variations.

Claims

1. A method for inducing Tetraena mongolica stem cells to form regenerated plants, characterized in that: The following steps are involved: Inducing and culturing Tetraena mongolica explants in a first induction culture medium to obtain cambium stem cells; transferring the cambium stem cells to a subculture culture medium for subculture to obtain Tetraena mongolica stem cells; suspending and culturing the Tetraena mongolica stem cells, and inducing callus tissue through a callus culture medium; The callus tissue is inoculated into a second induction medium containing a regeneration factor REF1 for culturing, and the regeneration factor REF1 promotes the gene transformation of the embryonic callus tissue, thereby differentiating the embryonic callus tissue; The embryonic callus is transferred to a third induction medium containing a regeneration factor REF1 for culturing, the regeneration factor REF1 is used to promote the differentiation of embryoids to obtain embryoids of Tetraena mongolica, the embryoids are inoculated into a bud proliferation medium for culturing to obtain Tetraena mongolica cluster buds, the Tetraena mongolica cluster buds are inoculated into a rooting medium for culturing to obtain Tetraena mongolica seedlings, i.e., Tetraena mongolica regenerated plants; In the second induction medium and the third induction medium, the concentration of the regeneration factor REF1 is 100 nM to 200 nM.

2. The method for inducing Tetraena mongolica stem cells to form regenerated plants according to claim 1, characterized in that: The first induction medium is an MS medium containing 30 g / L to 40 g / L sucrose, 500 mg / L to 700 mg / L hydrolyzed milk protein, 3 g / L to 4 g / L gellan gum, 0.5 mg / L to 1 mg / L 6-benzylaminopurine and 0.25 mg / L to 1 mg / L 2,4-dichlorophenoxyacetic acid; the pH value of the first induction medium is 5.75 to 5.85; The induction culture conditions are: temperature 25°C to 30°C, humidity 60% to 80%, and light 8h / d to 12h / d.

3. The method for inducing Tetraena mongolica stem cells to form regenerated plants according to claim 1, characterized in that: The subculture medium is an MS medium containing 30 g / L to 40 g / L sucrose, 500 mg / L to 700 mg / L hydrolyzed milk protein, 3 g / L to 4 g / L gellan gum, 0.1 mg / L to 1 mg / L 6-benzylaminopurine and 0.1 mg / L to 2 mg / L 2,4-dichlorophenoxyacetic acid, and the pH value of the subculture medium is 5.75 to 5.85; The culture conditions of the subculture are: temperature 25° C. to 30° C., humidity 60% to 80%, and light culture 8 h / d to 12 h / d.

4. The method of inducing Tetraena mongolica stem cells to form regenerated plants according to claim 1, characterized in that: The specific process of the suspension culture is as follows: Inoculate 50 g to 100 g of Tetraena mongolica stem cells per liter of suspension culture medium and culture continuously under light for 15 to 20 days; The suspension culture medium is an MS culture medium containing 10 g / L to 30 g / L sucrose, 500 mg / L to 700 mg / L hydrolyzed milk protein, 3 g / L to 4 g / L gellan gum, 0.1 mg / L to 3 mg / L 6-benzylaminopurine and 0.25 mg / L to 1 mg / L 2,4-dichlorophenoxyacetic acid, and the pH value of the suspension culture medium is 5.75 to 5.85; The conditions of the suspension culture are: temperature 25°C to 30°C, humidity 60% to 80%, and continuous illumination for 15d to 20d.

5. The method for inducing Tetraena mongolica stem cells to form regenerated plants according to claim 1, characterized in that: The callus culture medium is an MS culture medium containing 10 g / L to 30 g / L sucrose, 500 mg / L to 600 mg / L hydrolyzed milk protein, 3 g / L to 4 g / L gellan gum, 0.8 mg / L to 1 mg / L 6-benzylaminopurine, and 0.5 mg / L to 1 mg / L 2,4-dichlorophenoxyacetic acid, and the pH value of the callus culture medium is 5.75 to 5.85; The culture conditions of the callus culture medium are: temperature 25° C. to 30° C., humidity 60% to 80%, and light intensity 10 h / d to 12 h / d.

6. The method of inducing Tetraena mongolica stem cells to form regenerated plants according to claim 1, characterized in that: The second induction medium is an MS medium containing 30 g / L to 40 g / L sucrose, 500 g / L to 700 mg / L hydrolyzed milk protein, and 3 g / L to 4 g / L gellan gum, and the pH value of the second induction medium is 5.75 to 5.85; The culture conditions of the second induction culture medium are: temperature 25°C-30°C, humidity 60°C-80%, and light culture 12h / d.

7. The method for inducing Tetraena mongolica stem cells to form regenerated plants according to claim 1, characterized in that: The third induction medium is an MS medium containing 30 g / L to 40 g / L sucrose, 500 mg / L to 700 mg / L hydrolyzed milk protein, 3 g / L to 4 g / L gellan gum, 0.1 mg / L to 0.5 mg / L 6-benzylaminopurine and 0.25 mg / L to 0.3 mg / L 2,4-dichlorophenoxyacetic acid, and the pH value of the third induction medium is 5.75 to 5.85; The culture conditions of the third induction culture medium are: temperature 25° C. to 30° C., humidity 60% to 80%, and light culture 10 h / d to 12 h / d.

8. The method of inducing Tetraena mongolica stem cells to form regenerated plants according to claim 1, characterized in that: The bud proliferation medium is a B5 medium containing 30 g / L to 40 g / L sucrose, 500 mg / L to 700 mg / L hydrolyzed milk protein, 3.6 g / L to 4 g / L gellan gum, and 0.1 mg / L to 1 mg / L naphthylacetic acid, and the pH value of the bud proliferation medium is 5.75 to 5.85; The culture conditions of the bud proliferation culture medium are: temperature 25° C. to 30° C., humidity 60% to 80%, and light culture 8h / d to 12h / d.

9. The method for inducing Tetraena mongolica stem cells to form regenerated plants according to claim 1, characterized in that: The rooting medium is a B5 medium containing 30 g / L to 40 g / L sucrose, 500 mg / L to 700 mg / L hydrolyzed milk protein, 3.6 g / L to 4 g / L gellan gum, 0.1 mg / L to 1 mg / L 6-benzylaminopurine, 1 mg / L to 2 mg / L naphthaleneacetic acid, and 1 g / L to 1.5 g / L activated carbon, and the pH value of the rooting medium is 5.75 to 5.85; The culture conditions of the rooting culture medium are: temperature 25° C. to 30° C., humidity 60% to 80%, and light culture 10 h / d to 12 h / d.

10. The method of inducing Tetraena mongolica stem cells to form regenerated plants according to claim 1, characterized in that: The specific process of obtaining the Tetraena mongolica explant is as follows: Cut the Tetraena mongolica branches into small sections of 5 cm to 8 cm, sterilize, and then cut into slices with a thickness of 2 mm to 4 mm to obtain the Tetraena mongolica explants; The specific process of the sterilization is as follows: rinse with water for 0.5h to 2h, then soak in 10wt% hydrogen peroxide solution for 1min to 2min, soak in 75wt% ethanol solution for 1min to 2min, rinse with sterile water 2 to 3 times, soak in 0.05wt% mercuric chloride solution for 10min to 20min, soak in sterile water for 1min to 3min, and then rinse with sterile water 5 to 6 times.

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