A method for breeding seedlings of the characteristic southern medicinal herb Cycas revoluta for metabolomics analysis

Abstract

The present invention belongs to the field of plant cultivation technology, and particularly relates to a method for breeding seedlings of Hainan cycad, a characteristic southern medicine for metabolomics analysis. The breeding method comprises the following steps: (1) removing the middle seed coat and disinfecting and sterilizing the Hainan cycad fruit with a storage period of less than 2 years; (2) seed germination culture exosome and exosome selection: pinnate leaves are obtained after germination culture, and the pinnate leaves are used as exosomes; (3) adventitious bud induction culture; (4) subculture proliferation culture, and the subculture proliferation culture medium is an MS culture medium containing 6-benzylaminopurine 2.0-3.0 mg / L, naphthaleneacetic acid 0.1-0.3 mg / L, sucrose 18-20 g / L, and carrageenan 8-10 g / L; (5) rooting culture. The breeding method of the present invention requires less propagation material and a large proliferation coefficient. The 30-day differentiation bud proliferation coefficient exceeds 3.0, even reaching 3.3, and the number of new roots sprouting reaches 4.5.

Description

Technical Field

[0001] The invention belongs to the technical field of plant cultivation, and particularly relates to a method for breeding seedlings of Hainan Cycas, a characteristic southern medicinal material for metabolomics analysis. Background Art

[0002] Southern medicinal materials are traditional Chinese medicines mainly produced in tropical and subtropical regions. They are an important part of the traditional Chinese medicine industry and have distinct industrial characteristics such as a wide variety of varieties, large total economic output value, and strong regionality. In the past decade, the production of southern medicinal materials has developed rapidly, with increasing varieties, and significantly improved planting areas, total output, total output value, and benefits. Southern medicinal materials have become one of the important pillar industries for increasing agricultural efficiency and farmers' income in tropical areas.

[0003] Hainan Cycas (Cycas hainanensis CJChen), a species endemic to Hainan, has been included in the "National Key Protected Wild Plant List" (first batch), a national first-class protected plant, and is listed as an endangered plant by the International Union for Conservation of Nature and Natural Resources (IUCN).

[0004] Plant metabolomics is a technology for high-throughput, unbiased and comprehensive analysis of metabolites in plant extracts. The application of this technology to analyze the genetic structure and genetic diversity of Hainan Cycas is of great significance. However, since Hainan Cycas is dioecious, natural flowering and fruiting requires an appropriate environment. In addition, the distribution of male and female Hainan Cycas plants in nature is uneven. Therefore, there are currently three methods for Hainan Cycas reproduction: sowing, cutting and cutting, and breaking off tillers. These methods have high requirements for the growth of the mother plant and have limited breeding efficiency. In practical work, the sowing germination rate of Hainan Cycas is significantly lower than that of plants such as Cycas, Xianhu Cycas, and Panzhihua Cycas, which are also in the genus Cycas. Even by removing the mesopecten and soaking in chemicals, the sowing germination rate of Hainan Cycas with a short storage period and freshness is below 30%. At present, its wild population is facing the risk of extinction. It is difficult to breed seedlings in large quantities by cutting plant stems like Cycas, and it is also difficult to collect a large number of plants by breaking off tillers for metabolomics analysis.

[0005] Therefore, there is an urgent need to provide a new method for breeding seedlings of the southern medicinal herb Cycas Hainanensis, which can significantly improve the sowing germination rate and proliferation coefficient of Cycas Hainanensis. Summary of the invention

[0006] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes a method for breeding seedlings of a characteristic southern medicinal plant, Cycas revoluta, for metabolomics analysis. The breeding method of the present invention enables Cycas revoluta to have a high germination rate and a large 30-day proliferation coefficient.

[0007] The inventive concept of the present invention is to use the young pinnate leaves germinated by seeds as explants to induce the cultivation of adventitious buds for re-proliferation, and use a subculture proliferation medium with a specific composition in the process of subculture proliferation cultivation; differentiate clustered buds (also called bud clusters) and induce the root system of the monomers to become a large number of new Hainan Cycas plants. The technology of the present invention can promote the study of the genetic structure and genetic diversity of Hainan Cycas, and play an important role in the protection and development and utilization of its germplasm resources.

[0008] The breeding method of the present invention requires less propagation materials and has a large proliferation coefficient, and the proliferation coefficient of the differentiated buds in 30 days exceeds 3.0 and can reach 3.3. The present invention induces explants by treating and disinfecting the fruit of Hainan Cycas, inducing differentiated buds in a culture medium, and inducing root production according to demand through multiple proliferation and expansion, so as to achieve the purpose of rapid propagation of seedlings and then application, which is of great significance to the research on Hainan Cycas seedlings for metabolomics analysis and the protection of endangered plant resources.

[0009] The invention provides a method for breeding seedlings of the characteristic southern medicinal material Cycas hainanensis for metabolomics analysis.

[0010] Specifically, a method for breeding seedlings of Hainan Cycas, a characteristic southern medicinal herb for metabolomics analysis, comprises the following steps:

[0011] (1) taking the fruits of Cycas revoluta with a room temperature storage period of less than 2 years, removing the seed coat of the fruits of Cycas revoluta and performing disinfection and sterilization treatment;

[0012] (2) Seed germination culture explants and explant selection: Pinnate leaves are obtained after germination culture and used as explants;

[0013] (3) adventitious bud induction culture: implanting the explants into an adventitious bud induction medium for induction culture to obtain adventitious buds;

[0014] (4) Subculture proliferation culture: the adventitious buds of step (3) are cut into single buds with a scalpel for culture, and then the induced bud clusters are cut into single buds, the tops are cut off, and the buds are implanted in a subculture proliferation medium for culture, wherein the subculture proliferation medium is an MS medium containing 2.0-3.0 mg / L 6-BA (6-benzylaminopurine), 0.1-0.3 mg / L NAA (naphthaleneacetic acid), 18-20 g / L sucrose, and 8-10 g / L carrageenan;

[0015] (5) Rooting culture: From the bud cluster obtained through the subculture proliferation culture in step (4), a single bud is cut and implanted into a rooting medium for rooting culture.

[0016] Preferably, in step (1), the process of removing the middle seed coat is: using a nutcracker to crack the middle seed coat to take out the seeds.

[0017] Preferably, in step (1), the disinfection and sterilization treatment is as follows: placing the seeds with the middle seed coat removed in a container (e.g., a wide-mouth bottle), pouring 60-70% alcohol into the container and soaking for 8-10 seconds, then pouring sterile water into the container and rinsing for 2-5 times, soaking the rinsed seeds in 0.02% mass concentration of mercuric chloride on a clean bench for surface sterilization for 3-5 minutes, and rinsing with sterile water for 3-4 times, and then soaking and disinfecting with 2% mass fraction of sodium hypochlorite for 15-20 minutes, and rinsing with sterile water for 3-5 times, and slightly drying.

[0018] Preferably, in step (2), the process of seed germination culture is: the Hainan Cycas seeds sterilized in step (1) are implanted with the seed eyes facing upwards into the culture medium for germination, the implantation depth is 1.0-1.5 cm, and the seeds are transferred to the culture room for cultivation. The temperature during germination culture is 24-28°C, and the seeds are kept away from light and dark. The culture medium used for germination culture is MS culture medium containing 0.08-0.2 mg / L NAA (naphthaleneacetic acid), 0.1-0.2 mg / L IAA (indoleacetic acid), 4-6 ml / L ipiroxin, 8-12 g / L sucrose, and 8-12 g / L carrageenan.

[0019] Preferably, the culture medium used for germination culture is MS medium containing 0.1 mg / L NAA (naphthaleneacetic acid), 0.15 mg / L IAA (indoleacetic acid), 5 ml / L ipiroxin, 10 g / L sucrose, and 10 g / L carrageenan.

[0020] Preferably, the pH of the culture medium used for germination culture is 5.5-6.5, preferably 6.0.

[0021] Preferably, in step (2), the process of selecting the explants is as follows: after 15-18 days of germination culture, the seeds begin to germinate, and when the length of the sprouted pinnate leaves reaches 6-8 cm, they are cut into 2-3 cm segments with a scalpel as explants.

[0022] Preferably, in step (3), the culture temperature of the induced culture is 24-26°C, the illumination time is 7-8h / d, and the illumination intensity is 1200-1500lx.

[0023] Preferably, in step (3), the adventitious bud induction medium is an MS medium containing 0.25-1.0 mg / L 6-BA (6-benzylaminopurine), 0.1-0.3 mg / L NAA (naphthaleneacetic acid), 4-5 ml / L ipiroxin, 18-20 g / L sucrose, and 8-10 g / L carrageenan.

[0024] Preferably, in step (3), the pH of the adventitious bud induction medium is 6.0-6.8, preferably 6.5.

[0025] Preferably, in step (3), 9-10 days after the explants are implanted into the adventitious bud induction medium, the buds begin to grow, and 2-3 adventitious buds emerge from the leaf base in 28-30 days.

[0026] Preferably, in step (4), the adventitious buds of step (3) are cut into single buds with a scalpel for culture, and then the induced bud clusters are cut into single buds at intervals of 38-40 days, the tops are cut off, the buds are 1-2 cm long and implanted in subculture proliferation medium for culture.

[0027] Preferably, in step (4), the culture temperature is 26-28°C, the illumination time is 11-12h / d, and the illumination intensity is 1200-1500lx.

[0028] Preferably, in step (4), the pH of the secondary proliferation medium is 6.0-6.8, preferably 6.5.

[0029] Preferably, in step (4), after 8-10 days of implantation of a single bud in the subculture proliferation medium, small buds begin to appear, and the proliferation coefficient exceeds 3.0 after 28-30 days of implantation in the subculture proliferation medium. The next round of subculture proliferation is carried out every 30 days, using the same subculture proliferation medium.

[0030] Preferably, in step (5), from the bud cluster obtained by the subculture and proliferation culture in step (4), single buds longer than 1.5 cm are cut with a scalpel and implanted into a rooting medium, and buds shorter than 1.5 cm are continued to be subcultured and proliferated.

[0031] Preferably, in step (5), the temperature of the rooting culture is 26-28°C, the illumination time is 8-9h / d, and the illumination intensity is 1450-1500lx.

[0032] Preferably, in step (5), the rooting medium is a 1 / 2MS medium containing 0.15-0.2 mg / L IAA (indoleacetic acid), 0.5-0.6 mg / L NAA (naphthaleneacetic acid), 18-20 g / L sucrose, and 8-10 g / L carrageenan, with a pH value of 6.0-6.5.

[0033] Preferably, in step (5), rooting begins after 9-10 days of rooting culture, and the number of new roots sprouting reaches 4.5 after 29-30 days.

[0034] Application of the above breeding methods in metabolomics analysis.

[0035] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0036] (1) Using the young pinnate leaves germinated by seed germination as exosomes to induce the cultivation of adventitious buds for re-proliferation, and using a subculture proliferation medium with a specific composition in the process of subculture proliferation and cultivation; differentiating clustered buds (also called bud clusters) and inducing the root system of the monomers to become a large number of new Hainan Cycas plants. The present invention requires less propagation materials and has a large proliferation coefficient. The proliferation coefficient of the differentiated buds in 30 days exceeds 3.0, even reaches 3.3, and the number of new roots germinated exceeds 4.3, even reaches 4.5.

[0037] (2) The present invention innovatively uses the young pinnate leaves germinated from the seeds of Hainan Cycas as exoprogenits, which is beneficial to reducing the browning rate that occurs during the culture process; similarly, adding an appropriate amount of the highly effective anti-fouling fungicide Yiperon to the MS culture medium for seed culture exoprogenits and adventitious bud induction culture can significantly reduce the contamination rate.

[0038] (3) At present, there is no research on a method for breeding seedlings of Hainan Cycas, a special southern medicinal herb for metabolomics analysis, at home and abroad. The present invention can breed seedlings on a large scale in a short period of time and supply a large number of plants for metabolomics analysis and genetic engineering breeding research. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is the situation of rooting culture in Example 1;

[0040] Figure 2 This is the situation of the seedlings transplanted after rooting and culture in Example 1. DETAILED DESCRIPTION

[0041] In order to make the technical scheme of the present invention more clearly understood by those skilled in the art, the following embodiments are listed for illustration. It should be pointed out that the following embodiments do not limit the protection scope of the present invention.

[0042] Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial sources or by existing known methods.

[0043] 6-BA (6-benzylaminopurine) was purchased from Shanghai Yien Chemical Technology Co., Ltd., reagent grade, purity 99%, specification 1g / bottle.

[0044] NAA (naphthaleneacetic acid) was purchased from Shanghai Yien Chemical Technology Co., Ltd., reagent grade, purity 96%, specification 5g / bottle.

[0045] IAA (indoleacetic acid) was purchased from Shanghai Yien Chemical Technology Co., Ltd., reagent grade, purity 98%, specification 1 g / bottle.

[0046] Example 1

[0047] A method for breeding seedlings of Hainan cycad, a special southern medicinal herb for metabolomics analysis, comprises the following steps:

[0048] (1) Removal of the mesopecten and disinfection of Hainan Cycas fruits: Select Hainan Cycas fruits with a room temperature storage period of 0.5 years, wipe off the external dirt and impurities with alcohol cotton, and use a total of 50 fruits. Then use a nutcracker to crack the mesopecten and take out the seeds. Put the seeds with the mesopecten removed in a sterilized wide-mouth bottle, pour in 70% volume fraction alcohol and soak for 10 seconds, then pour a large amount of sterile water into the wide-mouth bottle to rinse twice to reduce the killing of plant cells by alcohol; soak the rinsed seeds in 0.02% mass concentration of mercuric chloride on a clean bench for 5 minutes to sterilize the surface and rinse with sterile water 4 times; then soak and disinfect in 2% mass fraction sodium hypochlorite solution for 10 minutes, and rinse with sterile water 5 times, and the seeds are slightly dried;

[0049] (2) Seed germination explant culture and explant selection: The Hainan Cycas seeds sterilized in step (1) were implanted into the culture medium with the seed eyes facing upward for germination, the implantation depth was 1.0-1.5 cm, and the seeds were transferred to the culture room for cultivation. The temperature during the germination period was controlled at 24-28° C. and the seeds were kept away from light and dark. The culture medium was MS medium containing 0.1 mg / L NAA, 0.15 mg / L IAA, 5 ml / L Yiperon, 10 g / L sucrose, and 10 g / L carrageenan, and the pH value was 6.0. After 15-18 days of germination culture, the seeds began to germinate. When the length of the pinnate leaves reached 7 cm, they were cut into 2 cm long sections with a scalpel as explants. The germination rate of the seeds was 85% and the contamination rate was 9.7%.

[0050] (3) Adventitious bud induction culture: The uncontaminated explants of step (2) were implanted into an adventitious bud induction medium for induction culture to obtain adventitious buds. The culture temperature was controlled at 24-26°C, the illumination time was 8 h / d, and the illumination intensity was controlled at 1200-1300 lx. The induction medium was an MS medium containing 0.5 mg / L 6-BA, 0.1 mg / L NAA, 5 ml / L iberolon, 20 g / L sucrose, and 10 g / L carrageenan, with a pH value of 6.5. Three leaf segments were inoculated into each culture bottle, which was sealed and placed in a culture room for induction culture. Ten days after the explants were implanted ("d" means "day"), the buds began to grow, and 2-5 adventitious buds were differentiated from the leaf base at 30 days. The contamination rate at this stage was 5.4%;

[0051] (4) Subculture proliferation culture: the newly germinated buds in step (3) are cut into single buds with a scalpel for culture, and then the induced bud clusters are cut into single buds at intervals of 30 days, the tops are cut off, and the single buds are 1-2 cm long and implanted in subculture proliferation medium for culture, the culture temperature is controlled at 26-28°C, the light duration is 9 h / d, the light intensity is 1200-1300 lx, the subculture proliferation medium is an MS medium containing 2.5 mg / L 6-BA, 0.2 mg / L NAA, 20 g / L sucrose, and 10 g / L carrageenan, and the pH value is 6.5, three new buds are inoculated into each culture bottle, sealed, and placed in a culture room for proliferation culture, small buds begin to appear after 10 days of culture, and the proliferation coefficient reaches 3.3 at 30 days; thereafter, the next round of subculture proliferation is carried out every 30 days, and the same subculture proliferation medium is used for culture;

[0052] (5) Rooting culture: A single bud with a length of more than 1.5 cm was cut with a scalpel from the bud clusters proliferated in step (4) and implanted into a rooting medium. Buds smaller than 1.5 cm were continued to be transferred to the proliferating culture. The culture temperature was controlled at 26-28° C., the light duration was 9 h / d, the light intensity was 1500 lx, the rooting medium was a 1 / 2 MS medium containing 0.2 mg / L IAA, 0.6 mg / L NAA, 20 g / L sucrose, and 10 g / L carrageenan, with a pH value of 6.5. Rooting began 10 days after culture, and the number of new roots sprouted after 30 days reached 4.5 (average value).

[0053] Figure 1 This is the situation of rooting culture in Example 1; Figure 2 This is the situation of the seedlings transplanted after rooting and culture in Example 1.

[0054] Example 2

[0055] A method for breeding seedlings of Hainan cycad, a special southern medicinal herb for metabolomics analysis, comprises the following steps:

[0056] (1) Removal of the seed coat and disinfection and sterilization of Hainan Cycas fruits: The selected fruits are Hainan Cycas fruits with a storage period of 1.5 years at room temperature, a total of 50 fruits, and the surface is sterilized for 3 minutes by soaking in 0.02% mass concentration of mercuric chloride in the disinfection and sterilization method, and the remaining operations are the same as in Example 1;

[0057] (2) Seed germination explant culture and explant selection: The operation was the same as in Example 1, and the seed germination rate was 76% and the contamination rate was 10.5%;

[0058] (3) Adventitious bud induction culture: The operation was the same as in Example 1. 2-4 adventitious buds were differentiated after 30 days, and the contamination rate was 7.4%;

[0059] (4) Subculture proliferation culture: The operation was the same as in Example 1, and the proliferation coefficient was 3.1 after 30 days;

[0060] (5) Rooting culture: The operation was the same as in Example 1. Rooting began after 10 days of culture, and the number of new roots sprouted reached 4.3 (average value) after 30 days.

[0061] Comparative Example 1

[0062] A method for breeding seedlings of Hainan cycad, a special southern medicinal herb for metabolomics analysis, comprises the following steps:

[0063] (1) Removal of the middle seed coat of the fruit of Cycas hainanensis and disinfection and sterilization: the operation is the same as in Example 1;

[0064] (2) Seed germination explant culture and explant selection: The difference between comparative example 1 and example 1 is that in step (2), the explants are selected from the germinated terminal buds, and the terminal buds are cut into bud blocks as explants using a scalpel. The remaining operations are the same as those in example 1. The seed germination rate is 82% and the contamination rate is 9.8%;

[0065] (3) Adventitious bud induction culture: The operation was the same as in Example 1. After 30 days, 1-2 adventitious buds were differentiated, and the contamination rate was 8.6%;

[0066] (4) Subculture proliferation culture: The operation was the same as in Example 1, and the proliferation coefficient was 2.3 after 30 days;

[0067] (5) Rooting culture: The operation was the same as in Example 1. Rooting began after 10 days of culture, and the number of new roots sprouted reached 4.3 (average value) after 30 days.

[0068] Comparative Example 2

[0069] A method for breeding seedlings of Hainan cycad, a special southern medicinal herb for metabolomics analysis, comprises the following steps:

[0070] (1) Removal of the middle seed coat of Hainan Cycas fruit and disinfection and sterilization: the operation is the same as in Example 2;

[0071] (2) Seed germination explant culture and explant selection: The difference between Comparative Example 2 and Example 2 is that in step (2), the explants are selected from the germinated terminal buds, and the terminal buds are cut into bud blocks as explants using a scalpel. The remaining operations are the same as those in Example 2. The seed germination rate is 76% and the contamination rate is 10.5%;

[0072] (3) Adventitious bud induction culture: The difference between Comparative Example 2 and Example 2 is that 5 ml / L of Yiperon was not added to the culture medium in step (3), and the rest of the operation was the same as in Example 2. 1-2 adventitious buds were differentiated in 30 days, and the contamination rate was 16.3%;

[0073] (4) Subculture proliferation culture: The operation was the same as in Example 2, and the proliferation coefficient was 1.3 after 30 days.

[0074] (5) Rooting culture: The operation was the same as in Example 2. Rooting began after 10 days of culture, and the number of new roots sprouted reached 3.0 (average value) after 30 days.

[0075] Comparative Example 3

[0076] A method for breeding seedlings of Hainan cycad, a special southern medicinal herb for metabolomics analysis, comprises the following steps:

[0077] (1) Removal of the middle seed coat and disinfection and sterilization of Hainan Cycas fruits: The fruits selected were Hainan Cycas fruits with a room temperature storage period of 2.5 years, and the remaining operations were the same as in Example 1.

[0078] (2) Seed germination explant culture and explant selection: The operation was the same as in Example 1, and the seed germination rate was 40% and the contamination rate was 65.5%;

[0079] (3) Adventitious bud induction culture: The operation was the same as in Example 1. After 30 days, 1-2 adventitious buds were differentiated, and the contamination rate was 8.2%;

[0080] (4) Subculture proliferation culture: the operation was the same as in Example 1, and the proliferation coefficient was 1.2 after 30 days;

[0081] (5) Rooting culture: The operation was the same as in Example 1. Rooting began after 10 days of culture, and the number of new roots sprouted reached 3.7 (average value) after 30 days.

[0082] Comparative Example 4

[0083] A method for breeding seedlings of Hainan cycad, a special southern medicinal herb for metabolomics analysis, comprises the following steps:

[0084] (1) Removal of the middle seed coat of the fruit of Cycas hainanensis and disinfection and sterilization: the operation is the same as in Example 1;

[0085] (2) Seed germination culture explants and explant selection: The difference between Comparative Example 4 and Example 1 is that the NAA in the MS culture medium is 0.2 mg / L and the IAA is 0.2 mg / L. The other compositions and operations are the same as those in Example 1. The seed germination rate is 84% ​​and the contamination rate is 9.7%.

[0086] (3) Adventitious bud induction culture: The difference between Comparative Example 4 and Example 1 is that the 6-BA in the MS medium is 2.0 mg / L and the NAA is 0.2 mg / L. The other compositions and operations are the same as those in Example 1. 3-6 adventitious buds are differentiated in 30 days, and the contamination rate is 5.6%;

[0087] (4) Subculture proliferation culture: The difference between Comparative Example 4 and Example 1 is that the 6-BA in the MS medium is 5.0 mg / L and the NAA is 0.2 mg / L. The other compositions and operations are the same as those in Example 1. The proliferation coefficient is 2.3 after 30 days.

[0088] (5) Rooting culture: The operation was the same as in Example 1. Rooting began 10 days after culture, and the number of new roots sprouted reached 4.4 (average value) on day 30.

[0089] Product effect testing

[0090] The experimental results shown in Table 1 below were obtained through Example 1, Example 2, and Comparative Examples 1-4.

[0091] Table 1

[0092]

[0093] As shown in Table 1, in Comparative Example 1, during the adventitious bud induction culture stage, the newly germinated apical buds were used as explants to induce adventitious buds. The number of adventitious buds differentiated in 30 days was 1-2, the contamination rate was 8.6%, and the highest proliferation coefficient in the proliferation stage of 30 days was only 2.3; in Comparative Example 2, the high-efficiency sterilizer Yiperon was not added to the adventitious bud induction culture medium, and its contamination rate was as high as 16.3%; in Comparative Example 3, due to the use of fruits with a storage period of more than 2 years for germination, its germination time was long, requiring 35-45 days, the germination rate was only 40%, and the contamination rate was as high as 65.5%. The subsequent operation was the same as that of Example 1, and its proliferation coefficient was only 1.2. It can be seen from the above data that the use of fruits with a storage period of less than 2 years, adding Yiperon to the culture medium for germination culture and adventitious bud induction can increase the germination rate, shorten the germination time, and significantly reduce the contamination rate. Similarly, young pinnate leaves were selected as explants, and the number of differentiated adventitious buds was large, and the subsequent proliferation coefficient was as high as 3.3.

[0094] It can be seen from Comparative Example 4 that adjusting the composition of the culture medium has a significant effect on the number of adventitious buds at 30 days and the proliferation coefficient at 30 days during adventitious bud induction culture.

[0095] The above experimental data show that the method for breeding Hainan Cycas seedlings for metabolomics analysis of the present invention can breed seedlings on a large scale in a short period of time and supply a large number of sterile plants for metabolomics analysis and genetic engineering breeding research.

Claims

1. A method for breeding seedlings of the characteristic southern medicinal herb Cycas hainanensis for metabolomics analysis, characterized in that: The following steps are involved: (1) taking the fruits of Cycas revoluta with a room temperature storage period of less than 2 years, removing the seed coat of the fruits of Cycas revoluta and performing disinfection and sterilization treatment; (2) Seed germination culture explants and explant selection: Pinnate leaves are obtained after germination culture and used as explants; (3) adventitious bud induction culture: implanting the explants into an adventitious bud induction medium for induction culture to obtain adventitious buds; (4) Subculture proliferation culture: the adventitious buds of step (3) are cut into single buds with a scalpel for culture, and then the induced bud clusters are cut into single buds, the tops are cut off, and the buds are implanted in a subculture proliferation medium for culture, wherein the subculture proliferation medium is an MS medium containing 2.0-3.0 mg / L 6-benzylaminopurine, 0.1-0.3 mg / L naphthaleneacetic acid, 18-20 g / L sucrose, and 8-10 g / L carrageenan; (5) Rooting culture: cutting a single bud from the bud cluster obtained by the subculture proliferation culture in step (4) and implanting it in a rooting medium for rooting culture; In the step (3), the adventitious bud induction medium is an MS medium containing 0.25-1.0 mg / L 6-benzylaminopurine, 0.1-0.3 mg / L naphthaleneacetic acid, 4-5 ml / L ipiroxin, 18-20 g / L sucrose, and 8-10 g / L carrageenan; In the step (5), the rooting medium is a 1 / 2MS medium containing 0.15-0.2 mg / L indoleacetic acid, 0.5-0.6 mg / L naphthaleneacetic acid, 18-20 g / L sucrose, and 8-10 g / L carrageenan, with a pH value of 6.0-6.

5.

2. The breeding method according to claim 1, characterized in that: In step (2), the process of seed germination culture is: the Hainan Cycas seeds sterilized in step (1) are implanted into the culture medium with the seed eyes facing upward for germination, the implantation depth is 1.0-1.5 cm, and the seeds are transferred to the culture room for cultivation. The temperature during germination culture is 24-28° C. and the seeds are kept away from light and dark. The culture medium used for germination culture is MS culture medium containing 0.08-0.2 mg / L of naphthaleneacetic acid, 0.1-0.2 mg / L of indoleacetic acid, 4-6 ml / L of ipirolon, 8-12 g / L of sucrose, and 8-12 g / L of carrageenan.

3. The breeding method according to claim 1, characterized in that: In step (2), the process of selecting explants is as follows: after 15-18 days of germination culture, the seeds begin to germinate, and when the length of the sprouted pinnate leaves reaches 6-8 cm, they are cut into 2-3 cm sections with a scalpel as explants.

4. The breeding method according to claim 1, characterized in that: In step (3), the culture temperature of the induced culture is 24-26°C, the illumination time is 7-8h / d, and the illumination intensity is 1200-1500lx.

5. The breeding method according to claim 1, characterized in that: In the step (3), 9-10 days after the explants are implanted into the adventitious bud induction medium, the buds begin to grow, and 2-3 adventitious buds are produced at the leaf base in 28-30 days.

6. The breeding method according to claim 1, characterized in that: In step (4), the adventitious buds of step (3) are cut into single buds with a scalpel for culture, and then the induced bud clusters are cut into single buds at intervals of 38-40 days, the tops are cut off, the buds are 1-2 cm long and implanted in subculture proliferation medium for culture.

7. The breeding method according to claim 1, characterized in that: In the step (4), the culture temperature is 26-28°C, the illumination time is 11-12h / d, and the illumination intensity is 1200-1500lx.

8. The breeding method according to claim 1, characterized in that: In the step (4), after 8-10 days of implantation of a single bud in the subculture proliferation medium, small buds begin to appear, and the proliferation coefficient exceeds 3.0 after 28-30 days of implantation in the subculture proliferation medium; the next round of subculture proliferation is carried out every 30 days, using the same subculture proliferation medium.

9. Use of the breeding method according to any one of claims 1 to 8 in metabolomics analysis.

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