Method for culturing and transplanting corn transgenic tissue culture seedlings

Abstract

The invention discloses a method for culturing and transplanting corn transgenic tissue culture seedlings. The method for culturing and transplanting the corn transgenic tissue culture seedlings comprises the steps of immature embryo inoculation, II-type embryonic callus induction, adventitious bud induction, multiplication culture, rooting culture, culture in soil, seedling hardening and transplanting. By adopting the culture and transplanting method provided by the invention, the Zun-dan No.1 corn is used as the explant, and the multiplication culture is carried out under the sterile condition, so that the multiplication is fast, the induction rate is high, the breeding time can be greatly shortened, a large number of tissue culture seedlings can be obtained in a short time, the breeding time is saved, the transgenic breeding process is accelerated, and the method is easy to implement, convenient to operate and suitable for industrial production. A genetic transformation system is easy to establish, technical reference can be provided for exogenous gene transformation and transgenic breeding application of Zun-single No.1 corn, new corn germplasm with beneficial genes can be formed, corn transgenic breeding requirements can be met, the maturing rate of tissue culture seedlings and the seed number of a single plant can be increased, and accordingly planting benefits are improved.

Description

Technical Field

[0001] The invention relates to the technical field of corn transgenic tissue culture regeneration, and in particular to a culture and transplanting method of corn transgenic cultured seedlings. Background Art

[0002] Corn is one of the main food and feed crops in my country, and is also an important industrial raw material. It occupies a very important position in the national economy. With the continuous development of animal husbandry and processing industry, the demand for corn is bound to increase further. In order to improve the yield and quality of corn planting, the study of its gene transformation has become one of the hot spots in plant molecular biology research at home and abroad. The industrialization of corn transgenic has brought changes to the development of the global corn seed industry and corn production methods, and has provided important guarantees for food security, ecological security and farmers' income increase. Since the inbred lines commonly used in corn transgenic are susceptible to infection, have poor resistance, are susceptible to diseases, have male and female imbalance, and have a long growth time in the genetic transformation tissue culture room, the transgenic corn seedlings are old, have poor adaptability, and have a low fruiting rate. The corn tissue culture seedlings obtained by transgenic technology have a low fruiting rate and harvested grain number, which seriously limits the development of corn functional genome and transgenic technology. Therefore, it is very important to improve the fruiting rate and the number of grains per plant of tissue culture seedlings.

[0003] Conventional breeding technology can no longer meet the society's urgent demand for new corn varieties. The rapidly developing plant genetic engineering technology can break the isolation between species, introduce exogenous excellent genes, and improve the genetic traits of species in a targeted manner, making up for the shortcomings of conventional breeding. Its application will surely greatly promote the breeding process of high-yield, high-quality, and high-resistant new varieties. Therefore, the genetic transformation research of corn has obvious application and theoretical significance. Zundan No. 1 is a grain-type corn hybrid with the external selection line R736-1 of Zunyi Academy of Agricultural Sciences as the female parent and the self-selected line Z335 as the male parent. It was approved by Guizhou Province in 2023, and the approval number is Qianshenyu 20230014. The present invention uses Zundan No. 1 corn as an explant, and after the exogenous gene is introduced, a new transgenic germplasm can be directly produced, thereby obtaining a new regeneration system, which can greatly shorten the breeding time and accelerate the transgenic breeding process. This can provide a technical reference for the exogenous gene transformation and transgenic breeding application of Zundan No. 1 corn, which is very necessary. Summary of the invention

[0004] The technical problem to be solved by the present invention is to solve the problems existing in the background technology, and provide a method for culturing and transplanting tissue culture seedlings. The Zundan No. 1 corn is used as an explant. Under sterile conditions, the tissue culture is carried out, and the proliferation is fast and the induction rate is high, which can greatly shorten the breeding time and obtain a large number of tissue culture seedlings in a short period of time. The method is easy to implement and convenient to operate, and it is easy to establish a genetic transformation system. It can provide a technical reference for exogenous gene transformation and transgenic breeding application of Zundan No. 1 corn. Specifically, it is a method for culturing and transplanting transgenic corn seedlings.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is: a method for culturing and transplanting transgenic corn seedlings, the culturing and transplanting method comprising the following steps: S1. Inoculation of young embryos and induction of type II embryonic callus Selecting healthy and vigorous Zundan No. 1 corn as explants, using immature embryos of Zundan No. 1 corn 15 days after pollination as explants, inoculating them into callus induction medium for induction, so as to produce type II embryonic callus; S2. Adventitious bud induction The type II embryonic callus obtained in step S1 is inoculated into an embryoid induction medium under a sterile environment, and cultured in a light incubator under the conditions of a culture temperature of 25±1° C., a light duration of 8 h / d, and a light intensity of 1500 to 2500 Lx to induce adventitious buds, produce green embryoids, and obtain regenerated plants; S3. Proliferation Culture The regenerated plants induced by adventitious buds in step S2 are cut off with a sterile scalpel to remove thin and weak roots, and 2 to 3 leaves are retained for each plant; they are inoculated into a proliferation medium under a sterile environment, and cultured in a light incubator under the conditions of a culture temperature of 25±1°C, a light duration of 14h / d, and a light intensity of 1500 to 2500Lx, so that the regenerated plants produce thick new roots, obtain dark green leaves, and form complete regenerated plants; S4. Rooting culture The regenerated plants cultured in step S3 are cut into individual plants using a sterile scalpel, and 3 to 4 leaves are retained for each plant; individual seedlings with good growth are selected and inoculated into rooting medium under a sterile environment, and cultured in a light incubator under the conditions of a temperature of 25±1°C, a light duration of 12h / d, and a light intensity of 2000 to 3000Lx, and robust rooted seedlings are obtained after one week of culture; S5. Cultivation in soil Transplant the rooted seedlings cultured in step S4 into nutrient soil, and culture them in a light incubator under the conditions of a temperature of 27±1°C, a light duration of 8h / d, and a light intensity of 3500-4500Lx for 10-15 days to allow the plants to grow slowly; S6. Hardening and transplanting The hardened plants obtained in step S5 are moved out of the light incubator together with the culture container, and placed in a room with normal room temperature. They are placed under room temperature for 7 to 10 days for hardening, and then moved to the field or plant growth room to allow them to grow normally and bear fruit.

[0006] Furthermore, using the method for culturing and transplanting a transgenic corn seedling described in the present invention, the specific operation method of inoculating young embryos and inducing type II embryonic callus in step S1 is: take young ears of Zundan No. 1 corn 15 days after pollination, peel off the husk, disinfect the surface with alcohol in a clean bench, cut off the endosperm part of the upper 1 / 2 grain, pick young embryos with a size of 2.0 mm, and inoculate them with the scutellum of the young embryos facing upward into callus induction medium, and culture them in the dark at a temperature of 30°C, and induce dense granular type II embryonic callus after 25 to 35 days.

[0007] Furthermore, a method for culturing and transplanting a transgenic corn seedling according to the present invention is adopted, wherein the callus induction medium in step S1 includes an MS medium, and 2,4-dichlorophenoxyacetic acid with a concentration of 0.02 to 0.05 mg / L, 6-benzylaminopurine with a concentration of 0.5 to 1.0 mg / L, and L-proline with a concentration of 150 to 200 mg / L are added to the MS medium, wherein the MS medium contains sucrose with a mass concentration of 3%, agar with a mass concentration of 7 g / L, and its pH value is adjusted to 5.8 to 6.0.

[0008] Furthermore, a method for culturing and transplanting a transgenic corn seedling described in the present invention is adopted, wherein the embryoid induction medium in step S2 includes an MS medium, and 6-benzylaminopurine with a concentration of 0.5 to 1.0 mg / L and naphthaleneacetic acid with a concentration of 0.05 to 0.1 are added to the MS medium, wherein the MS medium contains a sucrose mass concentration of 3%, an agar mass concentration of 7 g / L, and its pH value is adjusted to 5.8 to 6.0.

[0009] Furthermore, a method for culturing and transplanting a transgenic corn seedling according to the present invention is adopted, wherein the proliferation culture medium in step S3 includes an MS culture medium, and 6-furfurylaminopurine with a concentration of 0.5 to 1.0 mg / L, indolebutyric acid with a concentration of 0.05 to 0.1, and L-proline with a concentration of 200 to 250 mg / L are added to the MS culture medium, wherein the MS culture medium contains a sucrose mass concentration of 3%, an agar mass concentration of 7 g / L, and its pH value is adjusted to 5.8 to 6.0.

[0010] Furthermore, a method for culturing and transplanting transgenic corn seedlings according to the present invention is adopted, wherein the rooting medium in step S4 includes a 1 / 2MS medium, and indolebutyric acid with a concentration of 0.5 to 1 mg / L and 6-furfurylaminopurine with a concentration of 0.5 to 1.0 mg / L are added to the 1 / 2MS medium, wherein the MS medium contains a sucrose mass concentration of 1.5%, an agar mass concentration of 7 g / L, and its pH is adjusted to 5.8 to 6.0.

[0011] Furthermore, a cultivation and transplanting method for a transgenic corn seedling according to the present invention is adopted, and the nutrient soil in step S5 is a mixed preparation of humus soil, wood ash, chicken manure and vermiculite. The specific preparation method is: humus soil, wood ash, chicken manure and vermiculite are mixed evenly in a mass ratio of 1:1, methyl thiophanate with an effective ingredient content of 70% and copper calcium sulfate with an effective ingredient content of 80% are added at a ratio of 1 gram each per 20 liters of nutrient soil, the mixture is added and mixed evenly, water is added until it is kneaded into a ball and falls apart when the hand is loosened, and the mixture is covered with a plastic film at room temperature and allowed to stand; on the second day, 20 g of water-soluble biological organic fertilizer is added per 20 liters of nutrient soil, and the biological organic fertilizer is added and mixed evenly, and finally covered with a plastic film at room temperature, and after standing for 2-3 days, the mixture is placed in a plastic nutrient pot with water absorption holes.

[0012] Furthermore, in the method for culturing and transplanting transgenic corn seedlings described in the present invention, the biological organic fertilizer is selected from organic fertilizers with N+P2O5+K2O≥5%, organic matter≥45%, and beneficial microorganisms≥1 billion / gram.

[0013] Furthermore, in the method for culturing and transplanting transgenic corn seedlings described in the present invention, the specifications of the plastic nutrient pots are 15cm×15cm×20cm or 20cm×20cm×25cm.

[0014] Furthermore, in the tissue culture and rapid propagation method of dormant buds of Potamogeton kaki described in the present invention, the callus induction medium, embryoid induction medium, proliferation medium and rooting medium are all sterilized at 121° C. for 25 to 30 minutes.

[0015] The method for culturing and transplanting a transgenic corn seedling according to the present invention is adopted. The MS culture medium is a basic culture medium formula commonly used in plant tissue culture technology, has a high inorganic salt concentration, can ensure the mineral nutrition required for tissue growth, and can also accelerate the growth of callus tissue. It is a relatively stable ion balance solution, has a high nitrate content, and the quantity and proportion of its nutrients are appropriate, which can meet the nutritional and physiological needs of plant cells. Therefore, it has a relatively wide range of applications. Most plant tissue cultures use it as a basic culture medium for rapid propagation.

[0016] The 6-furfurylaminopurine is a kind of plant cytokinin and also a non-natural cytokinin. Its chemical name is 6-glycosylaminopurine (or N6-furanmethyladenine) and its molecular formula is C 10 H9N 5O It is insoluble in water, but soluble in strong acid, alkali and glacial acetic acid. In addition to promoting cell division, it also delays the senescence of detached leaves and cut flowers, induces bud differentiation and development, and increases stomatal aperture.

[0017] 6-Benzylaminopurine is a type of plant cytokinin that has the functions of inhibiting the decomposition of chlorophyll, nucleic acid and protein in plant leaves, keeping them green and preventing aging; and transporting amino acids, auxins and inorganic salts to the treated parts. It is widely used in agriculture, trees and horticultural crops at all stages from germination to harvest.

[0018] Naphthaleneacetic acid is a type of plant growth hormone that can promote cell division and expansion, induce the formation of adventitious roots to increase fruit setting, prevent fruit drop, change the ratio of female and male flowers, etc. It can enter the plant through the tender epidermis of leaves and branches, and seeds, and be transported to the whole plant with the nutrient flow.

[0019] Indolebutyric acid is a type of plant auxin, mainly used for rooting of cuttings. It can induce the formation of rhizogenes, promote cell differentiation and division, is beneficial to the generation of new roots and the differentiation of the vascular system, and promotes the formation of adventitious roots of cuttings. It is widely used in rooting of cuttings of trees and flowers.

[0020] Compared with the prior art, the method for culturing and transplanting corn transgenic tissue culture seedlings of the present invention has the following beneficial effects: using Zundan No. 1 corn as an explant, under sterile conditions, through young embryo inoculation and type II embryonic callus induction, adventitious bud induction, proliferation culture, rooting culture and soil culture, and finally hardening and transplanting, the proliferation is fast, the induction rate is high, the breeding time can be greatly shortened, a large number of tissue culture seedlings can be obtained in a short period of time, the breeding time is saved, and the transgenic breeding process is accelerated. The method is easy to implement, convenient to operate, and easy to establish a genetic transformation system. It can provide technical reference for exogenous gene transformation and transgenic breeding application of Zundan No. 1 corn, form new corn germplasm with beneficial genes, not only can meet the needs of corn transgenic breeding, but also can improve the fruit setting rate of tissue culture seedlings and the number of fruiting grains per plant, thereby improving the planting efficiency. DETAILED DESCRIPTION

[0021] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] The reagents used in the embodiments of the present invention, unless otherwise specified, can be commercially available to implement the present invention, and the technical solutions involved, unless otherwise specified, can adopt conventional techniques in the art. Example 1

[0023] A method for cultivating and transplanting transgenic corn seedlings, the method comprising the following steps: S1. Inoculation of young embryos and induction of type II embryonic callus Select the strong and healthy Zundan No. 1 corn as explant, take the young embryo of Zundan No. 1 corn 15 days after pollination as explant, inoculate in callus induction medium for induction, so that it produces type II embryonic callus, the specific operation method is: take the young ear of Zundan No. 1 corn 15 days after pollination, peel off the husk, disinfect the surface with alcohol in a clean bench, cut off the endosperm part of the upper 1 / 2 grain, pick the young embryo of 2.0 mm in size, make the young embryo shield upward inoculate in callus induction medium, culture in the dark at a temperature of 30 ℃, and induce dense granular type II after 25 days. Embryogenic callus; wherein the callus induction medium comprises MS medium, and 0.02 mg / L 2,4-dichlorophenoxyacetic acid, 0.5 mg / L 6-benzylaminopurine, and 150 mg / L L-proline are added to the MS medium, wherein the MS medium contains 3% sucrose and 7 g / L agar, and the pH value thereof is adjusted to 5.8-6.0; S2. Adventitious bud induction The type II embryonic callus obtained in step S1 is inoculated into an embryoid induction medium under a sterile environment, and cultured in a light incubator under the conditions of a culture temperature of 25±1°C, a light duration of 8h / d, and a light intensity of 1500Lx to induce adventitious buds, produce green embryoids, and obtain regenerated plants; wherein the embryoid induction medium includes an MS medium, and 6-benzylaminopurine with a concentration of 0.5mg / L and naphthaleneacetic acid with a concentration of 0.05 are added to the MS medium, wherein the MS medium contains sucrose with a mass concentration of 3%, agar with a mass concentration of 7g / L, and its pH value is adjusted to 5.8-6.0; S3. Proliferation Culture The regenerated plants induced by adventitious buds in step S2 are cut off with a sterile scalpel to remove the thin roots, and 2 leaves are retained for each plant; they are inoculated into a proliferation medium under a sterile environment, and cultured in a light incubator under the conditions of a culture temperature of 25±1°C, a light duration of 14h / d, and a light intensity of 1500Lx, so that the regenerated plants produce thick new roots, obtain dark green leaves, and form complete regenerated plants; wherein the proliferation medium includes an MS medium, and the MS medium is added with 6-furfurylaminopurine at a concentration of 0.5mg / L, indolebutyric acid at a concentration of 0.05, and L-proline at a concentration of 200mg / L, wherein the MS medium contains sucrose at a mass concentration of 3%, agar at a mass concentration of 7g / L, and its pH value is adjusted to 5.8-6.0; S4. Rooting culture The regenerated plants cultured in step S3 are cut into individual plants using a sterile scalpel, and 3 leaves are retained for each plant; individual seedlings with good growth are selected and inoculated into rooting medium under a sterile environment, and cultured in a light incubator under the conditions of a temperature of 25±1°C, a light duration of 12h / d, and a light intensity of 2000Lx, and robust rooted seedlings are obtained after one week of culture; wherein the rooting medium includes a 1 / 2MS medium, and indolebutyric acid with a concentration of 0.5mg / L and 6-furfurylaminopurine with a concentration of 0.5mg / L are added to the 1 / 2MS medium, wherein the MS medium contains a sucrose mass concentration of 1.5%, an agar mass concentration of 7g / L, and its pH is adjusted to 5.8-6.0.

[0024] S5. Cultivation in soil The rooted seedlings cultured in step S4 are transplanted into nutrient soil, and cultured in a light incubator under the conditions of a temperature of 27±1°C, a light duration of 8h / d, and a light intensity of 3500Lx for 10 days to slow down the growth of the plants; wherein the nutrient soil is a mixed preparation of humus soil, wood ash, chicken manure and vermiculite, and the specific preparation method is: humus soil, wood ash, chicken manure and vermiculite are mixed uniformly in a mass ratio of 1:1, and 1 gram of methyl thiophanate with an effective ingredient content of 70% and 1 gram of copper calcium sulfate with an effective ingredient content of 80% are added to every 20 liters of nutrient soil. The proportion of 20g of biological organic fertilizer dissolved in water is added to every 20 liters of nutrient soil, and the biological organic fertilizer is added and mixed evenly. Finally, the biological organic fertilizer is covered with a plastic film at room temperature, and the mixture is left to stand for 2-3 days. The mixture is then placed in a plastic nutrient pot with a size of 15cm×15cm×20cm and a water absorption hole. The biological organic fertilizer is selected from organic fertilizers with N+P2O5+K2O≥5%, organic matter≥45%, and beneficial microorganisms≥1 billion / gram. S6. Hardening and transplanting The hardened plants obtained in step S5 are moved out of the light incubator together with the culture container, placed in a room with normal room temperature, and hardened at room temperature for 7 days, and then moved to the field or plant growth room to allow them to grow normally; the final transplant survival rate reaches 97%. Example 2

[0025] A method for cultivating and transplanting transgenic corn seedlings, the method comprising the following steps: S1. Inoculation of young embryos and induction of type II embryonic callus The healthy and vigorous Zundan No. 1 corn was selected as the explant, and the immature embryos of Zundan No. 1 corn 15 days after pollination were used as the explants, and were inoculated into a callus induction medium for induction, so that it could produce type II embryonic callus; the specific operation method was as follows: the young ears of Zundan No. 1 corn 15 days after pollination were taken, the husks were peeled off, the surface was disinfected with alcohol in a clean bench, the endosperm part of the upper 1 / 2 grain was cut off, and the immature embryos with a size of 2.0 mm were selected, and the immature embryos were inoculated into the callus induction medium with the shield upward, and dark culture was carried out at a temperature of 30°C, and dense granular type II callus was induced after 28 days. Embryogenic callus; wherein the callus induction medium comprises MS medium, and 0.03 mg / L 2,4-dichlorophenoxyacetic acid, 0.8 mg / L 6-benzylaminopurine, and 180 mg / L L-proline are added to the MS medium, wherein the MS medium contains 3% sucrose and 7 g / L agar, and the pH value thereof is adjusted to 5.8-6.0; S2. Adventitious bud induction The type II embryonic callus obtained in step S1 is inoculated into an embryoid induction medium under a sterile environment, and cultured in a light incubator under the conditions of a culture temperature of 25±1°C, a light duration of 8h / d, and a light intensity of 2000Lx to induce adventitious buds, produce green embryoids, and obtain regenerated plants; wherein the embryoid induction medium includes an MS medium, and 6-benzylaminopurine with a concentration of 0.8mg / L and naphthaleneacetic acid with a concentration of 0.06 are added to the MS medium, wherein the MS medium contains sucrose with a mass concentration of 3%, agar with a mass concentration of 7g / L, and its pH value is adjusted to 5.8-6.0; S3. Proliferation Culture The regenerated plants induced by adventitious buds in step S2 are cut off with a sterile scalpel to remove the thin and weak roots, and 3 leaves are retained for each plant; they are inoculated into a proliferation medium under a sterile environment, and cultured in a light incubator under the conditions of a culture temperature of 25±1°C, a light duration of 14h / d, and a light intensity of 2000Lx, so that the regenerated plants produce thick new roots, obtain dark green leaves, and form complete regenerated plants; wherein the proliferation medium includes an MS medium, and the MS medium is added with 6-furfurylaminopurine at a concentration of 0.8mg / L, indolebutyric acid at a concentration of 0.06, and L-proline at a concentration of 230mg / L, wherein the MS medium contains sucrose at a mass concentration of 3%, agar at a mass concentration of 7g / L, and its pH value is adjusted to 5.8-6.0; S4. Rooting culture The regenerated plants cultured in step S3 are cut into individual plants using a sterile scalpel, and 3 leaves are retained for each plant; individual seedlings with good growth are selected and inoculated into a rooting medium under a sterile environment, and cultured in a light incubator under the conditions of a temperature of 25±1°C, a light duration of 12h / d, and a light intensity of 2500Lx, and robust rooted seedlings are obtained after one week of culture; wherein the rooting medium includes a 1 / 2MS medium, and indolebutyric acid with a concentration of 0.8mg / L and 6-furfurylaminopurine with a concentration of 0.8mg / L are added to the 1 / 2MS medium, wherein the MS medium contains a sucrose mass concentration of 1.5%, an agar mass concentration of 7g / L, and its pH is adjusted to 5.8-6.0; S5. Cultivation in soil The rooted seedlings cultured in step S4 are transplanted into nutrient soil, and cultured in a light incubator under the conditions of a temperature of 27±1°C, a light duration of 8h / d, and a light intensity of 4000Lx for 12 days to slow down the growth of the plants; wherein the nutrient soil is a mixed preparation of humus soil, wood ash, chicken manure and vermiculite, and the specific preparation method is as follows: humus soil, wood ash, chicken manure and vermiculite are mixed uniformly in a mass ratio of 1:1, and 1 % each of methyl thiophanate with an effective ingredient content of 70% and copper calcium sulfate with an effective ingredient content of 80% are added to every 20 liters of nutrient soil. grams, add and mix evenly, add water until it can be kneaded into a ball by hand and falls apart when loosened, cover with plastic film at room temperature and let stand; on the second day, add biological organic fertilizer at a ratio of 20g of water-soluble biological organic fertilizer per 20 liters of nutrient soil, add and mix evenly, finally cover with plastic film at room temperature, let stand for 2 days, and then put into a plastic nutrient pot with a specification of 20cm×20cm×25cm and water absorption holes; the biological organic fertilizer is selected from organic fertilizers with N+P2O5+K2O≥5%, organic matter≥45%, and beneficial microorganisms≥1 billion / gram.

[0026] S6. Hardening and transplanting The hardened plants obtained in step S5 are moved out of the light incubator together with the culture container, placed in a room with normal room temperature, and hardened at room temperature for 8 days. They are then moved to the field or plant growth room to allow them to grow normally and bear fruit. The final transplant survival rate reaches 98%. Example 3

[0027] The present embodiment provides a method for culturing and transplanting transgenic corn seedlings, the method comprising the following steps: S1. Inoculation of young embryos and induction of type II embryonic callus The robust Zundan No. 1 corn was selected as an explant, and the immature embryos of Zundan No. 1 corn 15 days after pollination were used as explants, and inoculated into a callus induction medium for induction, so that it produced type II embryonic callus; the specific operation method was as follows: the young ears of Zundan No. 1 corn 15 days after pollination were taken, the husks were peeled off, the surface was disinfected with alcohol in a clean bench, the endosperm part of the upper 1 / 2 grain was cut off, and the immature embryos with a size of 2.0 mm were selected, and the scutellum of the immature embryos was inoculated upward into the callus induction medium, and dark culture was carried out at a temperature of 30°C, and dense granular type II callus was induced after 35 days. Embryogenic callus; wherein the callus induction medium comprises MS medium, and 0.05 mg / L 2,4-dichlorophenoxyacetic acid, 1.0 mg / L 6-benzylaminopurine, and 200 mg / L L-proline are added to the MS medium, wherein the MS medium contains 3% sucrose and 7 g / L agar, and the pH value thereof is adjusted to 5.8-6.0; S2. Adventitious bud induction The type II embryonic callus obtained in step S1 is inoculated into an embryoid induction medium under a sterile environment, and cultured in a light incubator under the conditions of a culture temperature of 25±1°C, a light duration of 8h / d, and a light intensity of 2500Lx to induce adventitious buds, produce green embryoids, and obtain regenerated plants; wherein the embryoid induction medium includes an MS medium, and 6-benzylaminopurine with a concentration of 1.0mg / L and naphthaleneacetic acid with a concentration of 0.1 are added to the MS medium, wherein the MS medium contains sucrose with a mass concentration of 3%, agar with a mass concentration of 7g / L, and its pH value is adjusted to 5.8-6.0; S3. Proliferation Culture The regenerated plants induced by adventitious buds in step S2 are cut off with a sterile scalpel to remove the thin and weak roots, and 3 leaves are retained for each plant; they are inoculated into a proliferation medium under a sterile environment, and cultured in a light incubator under the conditions of a culture temperature of 25±1°C, a light duration of 14h / d, and a light intensity of 2500Lx, so that the regenerated plants produce thick new roots, obtain dark green leaves, and form complete regenerated plants; wherein the proliferation medium includes an MS medium, and the MS medium is added with 6-furfurylaminopurine at a concentration of 1.0mg / L, indolebutyric acid at a concentration of 0.1, and L-proline at a concentration of 250mg / L, wherein the MS medium contains sucrose at a mass concentration of 3%, agar at a mass concentration of 7g / L, and its pH value is adjusted to 5.8-6.0; S4. Rooting culture The regenerated plants cultured in step S3 are cut into individual plants using a sterile scalpel, and 4 leaves are retained for each plant; the individual seedlings with good growth are selected and inoculated into a rooting medium under a sterile environment, and cultured in a light incubator under the conditions of a temperature of 25±1°C, a light duration of 12h / d, and a light intensity of 3000Lx, and robust rooted seedlings are obtained after one week of culture; wherein the rooting medium includes a 1 / 2MS medium, and indolebutyric acid with a concentration of 1mg / L and 6-furfurylaminopurine with a concentration of 1.0mg / L are added to the 1 / 2MS medium, wherein the MS medium contains a sucrose mass concentration of 1.5%, an agar mass concentration of 7g / L, and its pH is adjusted to 5.8-6.0; S5. Cultivation in soil The rooted seedlings cultured in step S4 are transplanted into nutrient soil, and cultured in a light incubator under the conditions of a temperature of 27±1°C, a light duration of 8h / d, and a light intensity of 4500Lx for 15 days to slow down the growth of the plants; wherein the nutrient soil is a mixed preparation of humus soil, wood ash, chicken manure and vermiculite, and the specific preparation method is as follows: humus soil, wood ash, chicken manure and vermiculite are mixed uniformly in a mass ratio of 1:1, and 1 % each of methyl thiophanate with an effective ingredient content of 70% and copper calcium sulfate with an effective ingredient content of 80% are added to every 20 liters of nutrient soil. The mixture is mixed with water and then mixed evenly, and water is added until it is kneaded into a ball and falls apart when the hand is loosened, and the mixture is covered with a plastic film and allowed to stand at room temperature; on the second day, 20 g of bio-organic fertilizer dissolved in water is added to every 20 liters of nutrient soil, and the bio-organic fertilizer is added and mixed evenly, and finally covered with a plastic film at room temperature, and after standing for 3 days, the bio-organic fertilizer is placed in a plastic nutrient pot with a size of 20 cm × 20 cm × 25 cm and a water absorption hole; the bio-organic fertilizer is selected to have N+P2O5+K2O≥5%, organic matter≥45%, and beneficial microorganisms≥1 billion / gram; S6. Hardening and transplanting The hardened plants obtained in step S5 are moved out of the light incubator together with the culture container, placed in a room with normal room temperature, and hardened at room temperature for 10 days. They are then moved to the field or plant growth room to allow them to grow normally and bear fruit. The final transplant survival rate reaches 97%.

[0028] In the method for culturing and transplanting transgenic corn seedlings of the present invention, the callus induction medium, embryoid induction medium, proliferation medium and rooting medium described in Examples 1 to 3 are sterilized at 121° C. for 25 to 30 minutes.

[0029] In order to illustrate the cultivation and transplanting method of the present invention, Zundan No. 1 corn was used as an explant. Under sterile conditions, tissue culture was performed. In three embodiments, its growth was judged from two aspects: proliferation multiples and rhizome multiples. The specific situation is shown in Table 1.

[0030] Table 1 shows the growth conditions of Zundan No. 1 corn in three embodiments of the present invention.

[0031]

[0032] It can be seen that by using Zundan No. 1 corn as an explant, through proliferation culture under sterile conditions, the breeding time can be greatly shortened, a large number of tissue culture seedlings can be obtained in a short period of time, breeding time can be saved, and the process of transgenic breeding can be accelerated. The method is easy to implement, convenient to operate, and easy to establish a genetic transformation system. It can provide technical reference for exogenous gene transformation and transgenic breeding application of Zundan No. 1 corn, forming a new corn germplasm with beneficial genes, which can not only meet the needs of corn transgenic breeding, but also increase the fruit set rate of tissue culture seedlings and the number of fruit grains per plant, thereby improving the planting efficiency.

[0033] The protection scope of the present invention is not limited to the technical solutions disclosed in the specific implementation methods. The above description is only a preferred implementation method of the present invention and does not limit the present invention. Any minor modifications, equivalent substitutions and improvements made according to the technical solutions of the present invention should be included in the protection scope of the technical solutions of the present invention.

Claims

1. A method for cultivating and transplanting transgenic corn seedlings, characterized in that: The culture and transplanting method comprises the following steps: S1. Inoculation of young embryos and induction of type II embryonic callus Selecting healthy and vigorous Zundan No. 1 corn as explants, using immature embryos of Zundan No. 1 corn 15 days after pollination as explants, inoculating them into callus induction medium for induction, so as to produce type II embryonic callus; S2. Adventitious bud induction The type II embryonic callus obtained in step S1 is inoculated into an embryoid induction medium under a sterile environment, and cultured in a light incubator under the conditions of a culture temperature of 25±1° C., a light duration of 8 h / d, and a light intensity of 1500 to 2500 Lx to induce adventitious buds, produce green embryoids, and obtain regenerated plants; S3. Proliferation Culture The regenerated plants induced by adventitious buds in step S2 are cut off with a sterile scalpel to remove thin and weak roots, and 2 to 3 leaves are retained for each plant; they are inoculated into a proliferation medium under a sterile environment, and cultured in a light incubator under the conditions of a culture temperature of 25±1°C, a light duration of 14h / d, and a light intensity of 1500 to 2500Lx, so that the regenerated plants produce thick new roots, obtain dark green leaves, and form complete regenerated plants; S4. Rooting culture The regenerated plants cultured in step S3 are cut into individual plants using a sterile scalpel, and 3 to 4 leaves are retained for each plant; individual seedlings with good growth are selected and inoculated into rooting medium under a sterile environment, and cultured in a light incubator under the conditions of a temperature of 25±1°C, a light duration of 12h / d, and a light intensity of 2000 to 3000Lx, and robust rooted seedlings are obtained after one week of culture; S5. Cultivation in soil Transplant the rooted seedlings cultured in step S4 into nutrient soil, and culture them in a light incubator under the conditions of a temperature of 27±1°C, a light duration of 8h / d, and a light intensity of 3500-4500Lx for 10-15 days to allow the plants to grow slowly; S6. Hardening and transplanting The hardened plants obtained in step S5 are moved out of the light incubator together with the culture container, and placed in a room with normal room temperature. They are placed under room temperature for 7 to 10 days for hardening, and then moved to the field or plant growth room to allow them to grow normally and bear fruit.

2. The method for cultivating and transplanting transgenic corn seedlings according to claim 1, characterized in that: The specific operation method of the young embryo inoculation and type II embryonic callus induction in step S1 is as follows: take the young ears of Zundan No. 1 corn 15 days after pollination, peel off the husk, disinfect the surface with alcohol in a clean bench, cut off the endosperm part of the upper 1 / 2 grain, pick the young embryo with a size of 2.0 mm, inoculate the young embryo with the scutellum facing upward into the callus induction medium, culture in the dark at a temperature of 30°C, and induce dense granular type II embryonic callus after 25 to 35 days.

3. The method for cultivating and transplanting transgenic corn seedlings according to claim 1, characterized in that: The callus induction medium in step S1 includes MS medium, and 2,4-dichlorophenoxyacetic acid with a concentration of 0.02-0.05 mg / L, 6-benzylaminopurine with a concentration of 0.5-1.0 mg / L, and L-proline with a concentration of 150-200 mg / L are added to the MS medium, wherein the MS medium contains sucrose with a mass concentration of 3%, agar with a mass concentration of 7 g / L, and its pH value is adjusted to 5.8-6.

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4. The method for cultivating and transplanting transgenic corn seedlings according to claim 1, characterized in that: The embryoid induction medium in step S2 includes MS medium, and 6-benzylaminopurine with a concentration of 0.5-1.0 mg / L and naphthaleneacetic acid with a concentration of 0.05-0.1 are added to the MS medium, wherein the MS medium contains sucrose with a mass concentration of 3%, agar with a mass concentration of 7 g / L, and its pH value is adjusted to 5.8-6.

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5. The method for cultivating and transplanting transgenic corn seedlings according to claim 1, characterized in that: The proliferation medium in step S3 includes MS medium, and 6-furfurylaminopurine with a concentration of 0.5-1.0 mg / L, indolebutyric acid with a concentration of 0.05-0.1, and L-proline with a concentration of 200-250 mg / L are added to the MS medium, wherein the MS medium contains sucrose with a mass concentration of 3%, agar with a mass concentration of 7 g / L, and its pH value is adjusted to 5.8-6.

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6. The method for cultivating and transplanting transgenic corn seedlings according to claim 1, characterized in that: The rooting medium in step S4 includes 1 / 2MS medium, and indolebutyric acid with a concentration of 0.5-1 mg / L and 6-furfurylaminopurine with a concentration of 0.5-1.0 mg / L are added to the 1 / 2MS medium, wherein the MS medium contains sucrose with a mass concentration of 1.5%, agar with a mass concentration of 7 g / L, and its pH is adjusted to 5.8-6.

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7. The method for cultivating and transplanting transgenic corn seedlings according to claim 1, characterized in that: The nutrient soil in step S5 is a mixed preparation of humus soil, wood ash, chicken manure and vermiculite. The specific preparation method is: humus soil, wood ash, chicken manure and vermiculite are mixed evenly in a mass ratio of 1:1, methyl thiophanate with an effective ingredient content of 70% and copper calcium sulfate with an effective ingredient content of 80% are added in a ratio of 1 gram each per 20 liters of nutrient soil, and the mixture is added and mixed evenly, water is added until it can be kneaded into a ball and falls apart when the hand is loosened, and the mixture is covered with a plastic film at room temperature and allowed to stand; on the second day, 20 g of water-soluble biological organic fertilizer is added per 20 liters of nutrient soil, and the biological organic fertilizer is added and mixed evenly, and finally, the mixture is covered with a plastic film at room temperature, and after standing for 2-3 days, the mixture is placed in a plastic nutrient pot with water absorption holes.

8. The method for cultivating and transplanting transgenic corn seedlings according to claim 7, characterized in that: The bio-organic fertilizer is selected from organic fertilizers with N+P2O5+K2O≥5%, organic matter≥45%, and beneficial microorganisms≥1 billion / gram.

9. The method for cultivating and transplanting transgenic corn seedlings according to claim 7, characterized in that: The specifications of the plastic nutrient pot are 15cm×15cm×20cm or 20cm×20cm×25cm.

10. The method for tissue culture and rapid propagation of dormant buds of Potamogeton oleifera according to claim 1, characterized in that: The callus induction medium, embryoid induction medium, proliferation medium and rooting medium are all sterilized at 121° C. for 25 to 30 minutes.

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