Soybean in-vitro living body circulating breeding method

Abstract

According to the method, embryo tips of soybean seeds are taken as initial explants, and the soybean in-vitro living body circulating breeding regulation and control method is established through circulation of the steps of sterile germination, adventitious bud induction, adventitious bud elongation, adventitious bud rooting, in-vitro seedling strengthening, flowering induction, in-vitro hybridization, seed setting regulation and control, acclimatization and transplantation, in-vitro seed setting, sterile germination and the like. According to the method, the high-activity soybean seeds can be produced under the in-vitro condition, the soybean breeding period is greatly shortened, the method becomes a brand new soybean breeding way, the way is free of seasonal limitation, all-weather breeding is achieved, and technical support is provided for soybean molecular breeding and seedling breeding thereof.

Description

Technical Field

[0001] The invention belongs to the field of plant tissue culture and biotechnology, and in particular relates to a soybean in vitro living cycle breeding regulation method. Background Art

[0002] Soybean (Glycine max (L.) Merr) is an important economic crop and the world's largest oil crop. It is a good source of nutrients such as high-quality protein, dietary fiber, essential fatty acids, vitamins and minerals (Guo Shuntang, 2023). Soybean is widely used in the food / feed industry, and often causes huge fluctuations in the national economy by affecting the feed industry. Therefore, the soybean industry is a strategic industry with important influence in my country's national economy (Guo Shuntang, 2023). Current research on soybeans focuses on increasing yields, optimizing varieties, strengthening industry resilience and technological innovation, aiming to promote the high-quality development of the soybean industry. However, due to the influence of various factors such as history, economy, resources and environment, my country's soybean industry still has problems such as long-term difficulty in expanding the scale of planting, low influence on international pricing power, insufficient bargaining power for international freight, and weak industrial competitiveness (Bian Jing, 2023). It is very important to focus on strengthening the research and development and promotion and application of soybean production technology.

[0003] With the gradual implementation of Japan's plan to discharge nuclear wastewater into the sea, it poses a huge threat to the marine environment. Radioactive substances may enter the human body through marine fish and other foods. The international community generally expressed concern and opposition to Japan's nuclear wastewater discharge plan. The General Administration of Customs of China has also decided to suspend the import of aquatic products originating from Japan from August 24, 2023 to prevent the risk of radioactive contamination. Animal protein consumption has been affected by the discharge of nuclear wastewater from Japan, and countries around the world have increased their dependence on plant protein. Soybean tissue protein is not only low-priced and easy to obtain, but also has superior nutritional value. It exhibits excellent gelling, water retention and other food processing properties. It is recognized as an excellent choice for making plant protein meat (Zhang F, 2021; Zeng Yan, 2021). Since the industrialization of human society in the 18th century, climate warming caused by greenhouse gases has become a serious issue facing mankind. China is a sensitive area and a significant impact area of ​​global climate change, and the warming rate is significantly higher than the global average during the same period (Pan Shiqiu, 2022). Studies have shown that China's warming trend in the past 50-60 years is 0.23℃ / 10a. Both the total warming and the warming trend in the past century are higher than the global average (Ding Yihui, 2016). Temperature is the most sensitive climate factor for crop growth and development. This asynchrony between temperature change and crop growth and development will inevitably affect crop yield and quality. As China's main cash crop, soybeans have a large demand and a wide planting area. Their yield formation is directly affected by temperature and soil type (Salem, 2007). Temperature changes have an important impact on the growth and development of soybeans. Studies have shown that the optimal growth temperature for soybeans is 15-22℃ during germination and maturity, and 20-25℃ during flowering. When the ambient temperature is higher than 28℃, it will affect the normal development process of soybeans (Xu Jiaqi, 2017; Djanaguiraman, 2010). Compared with the vegetative growth stage, soybeans are more sensitive to temperature changes during the reproductive growth stage. When the ambient temperature reaches 33-40℃, it will seriously restrict the growth process of soybean plants and cause stunted development (Reddy, 2007). Temperature changes have greatly affected the distribution, growth, yield and quality of soybeans (Fhuzar, 2010; Liu X, 2008). Crop model studies have shown that soybean yield is negatively correlated with the temperature rise during its growth period. Climate warming will reduce soybean yield, and for every 1°C increase in temperature, soybean yield will decrease by an average of 17% (Lobell, 2008; Mall, 2004; Kucharik, 2008; Lobell, 2003). It is crucial to cultivate new soybean varieties that can withstand extreme climates. At present, there are many ways to breed soybeans, such as molecular breeding, introduction, hybrid breeding, cyclic breeding, systematic breeding, and radiation-induced mutagenesis breeding (Cui Shaobin, 2024). Although traditional hybrid breeding methods have achieved certain results in soybean breeding, they are inefficient, have a long cycle, and are restricted by seasons and the environment.With the continuous development of science and technology, molecular breeding technology has gradually been applied to soybean breeding and has achieved some results, but there is still a large gap compared with developed countries, which needs to be further studied. Flowering in a bottle technology can speed up the breeding process. Through 1-2 culture cycles, it can artificially promote flowering and fruiting in the bottle. According to the flowering or fruiting situation, individuals with excellent traits can be selected purposefully, which can reduce the workload of a large number of cultivation and make breeding work more targeted (Wu Gaoqiong, 2019). Moreover, using tissue culture technology to make soybeans fruiting in a bottle can also break the natural conditions that restrict soybean growth, and soybean seeds and breeding materials can be obtained all year round.

[0004] References

[0005] [1] Guo Shuntang, Xu Jingting. 2023. Development status and existing problems of soybean food industry in my country. Journal of Food Science and Technology, 41(03): 1-8.

[0006] [2] Bian Jing. Main problems and countermeasures facing the high-quality development of my country's soybean industry [J]. China Economic and Trade Guide, 2023, (06): 57-60. [3] Pan Shiqiu. Response of soybean photosynthetic characteristics and yield quality to warming [D]. Nanjing University of Information Science and Technology, 2022.

[0007] [4] Ding Yihui, Wang Huijun. New understanding of scientific issues on climate change in China over the past century[J]. Chinese Science Bulletin, 2016, 61(10): 1029-1041.

[0008] [5]Salem MA,Kakani VG,Koti S,et al.Pollen-based screening of soybean genotypes for high temperatures[J].Crop science,2007,47(1):219-231.

[0009] [6]Xu Jiaqi, Guo Lifeng, Yin Shiping, et al. Study on quantitative indicators of suitable temperature and precipitation for soybeans at different growth stages in Heilongjiang Province[J]. Journal of Northeast Agricultural University, 2017, 48(8): 33-44;

[0010] [7]Djanaguiraman M,Prasad P V.Ethylene production under hightemperature stress causes premature leaf senescence in soybean[J].Functionalplant biology,2010,37(11):1071-1084.

[0011] [8]Reddy K R,Kakani V G.Screening capsicum species of differentorigins for high temperature tolerance by in vitro pollen germination andpollen tube length[J].Scientia horticulturae,2007,112(2):130-135.

[0012] [9]Thuzar M,Puteh A B,Abdullah N A,et al.The effects of temperaturestress on the quality and yield of soybean[J].Journal of agriculturalscience,2010,2(1):172-179.

[0013]

[10] Liu X,Jin J,Wang G H,et al.Soybean yield physiology anddevelopment of high-yielding practices in northeast China[J].Field cropsresearch,2008,105(3):157-171.

[0014]

[11] Lobell D B,Burke M B,Tebaldi C,et al.Prioritizing climate changeadaptation needs for food security in 2030[J].Science,2008,319(5863):607-610.

[0015]

[12] Mall R K,Lal M,Bhatia V S,et al.Mitigating climate change impacton soybean productivity in India:a simulation study[J].Agricultural andforest meteorology,2004,121(1-2):113-125.

[0016]

[13] Kucharik CJ, Serbin S P. Impacts of recent climate change on Wisconsin corn and soybean yield trends[J]. Environmental research letters, 2008, 3(3):034003.

[0017]

[14] Lobell DB, Asner G P. Climate and management contributions to recent trends in US agricultural yields[J].Science,2003,299(5609):1032-1032.

[0018]

[15] Cui Shaobin, Yu Xiaoguang, Chen Xiangjin, et al. Research progress in combining conventional soybean breeding and molecular breeding[J]. Hebei Agriculture, 2024, (01): 45-46.

[0019]

[16] Wu Gaoqiong. Analysis of wild parents of ancient Chinese roses based on SSR molecular markers[D]. Yunnan University, 2019. Summary of the invention

[0020] The present invention uses full and healthy soybean seeds as starting explants, and induces soybean flowering in a bottle to produce seeds through a cycle of sterile germination, adventitious bud induction, adventitious bud elongation, adventitious bud rooting, in vitro seedling strengthening, flowering induction, in vitro hybridization, fruiting regulation, seedling hardening and transplanting, in vitro fruiting, sterile germination and the like, thereby establishing a soybean in vitro living cycle breeding regulation system, satisfying the soybean growth conditions in nature so that the soybean can grow without being affected by time and space factors, providing materials for test tube hybridization, and accelerating the breeding cycle; the soybean seeds produced at the same time can be used as starting materials for tissue culture, and provide excellent materials for soybean in vitro regeneration and gene editing.

[0021] The technical solution provided by the present invention is as follows:

[0022] A method for in vitro fruiting of soybeans, characterized in that the following steps are carried out in a culture container such as a culture bottle:

[0023] S1. Aseptic germination of parent soybean seeds: Select full, disease-free and insect-free soybean seeds, sterilize them, and then inoculate them into MS medium until seedlings grow;

[0024] S2 adventitious bud induction: transfer the embryo tips of the soybean germinated in step S1 to MS+1-5mg / L 6-BA medium for 3-10 days, and then transfer to MS+1-3mg / L 6-BA+0.1-0.3mg / L NAA to induce adventitious buds;

[0025] S3 adventitious bud elongation: the adventitious buds induced in step S2 are inoculated in an adventitious bud elongation induction medium and cultured until sprouts are produced, wherein the adventitious bud elongation induction medium is a basic medium of MS medium supplemented with 0.1-0.5 mg / L 6-BA and 0.01-0.05 mg / L NAA;

[0026] S4 adventitious bud rooting: the seedlings in step S3 were transferred to 1 / 2MS + 0.5-1.5mg / L IBA + 0.1-0.6mg / L NAA + 0.05-0.2mg / L ethephon medium for rooting induction;

[0027] S5 in vitro seedling cultivation: the seedlings from step S4 are transferred to a medium of MS + 0.1-1 mg / L 6-BA + 0.01-0.1 mg / L NAA or 2MS + 0.1-1 mg / L 6-BA + 0.01-0.1 mg / L NAA for seedling cultivation;

[0028] S6 in vitro flowering induction: when the seedlings in step S5 grow to the same height as the culture container, 0.4-1.25 g / L KH2PO4 is added to the culture medium and cultured until buds appear;

[0029] S7 Artificial pollination: In a clean environment, take soybean pollen to artificially pollinate the pistils of unopened flowers in the culture container, remove the unopened buds other than the pollinated flowers, mark the artificially pollinated soybean plants accordingly, and continue to culture;

[0030] S8 Fruiting regulation: After the soybeans in step S7 have formed pods, 0.1-1.0 g / L KNO3 is added to the culture medium until mature seeds are obtained.

[0031] Preferably,

[0032] In step S1, full and healthy soybean seeds are soaked in clean water for 3-6 hours, then treated with ethanol, soaked in sodium hypochlorite solution, and then washed with sterile water; preferably, after soaking in clean water for 5 hours, they are placed in 75% ethanol for 30 seconds, soaked in 20% sodium hypochlorite solution for 12-20 minutes, and washed with sterile water 3-5 times.

[0033] In step S2, the culture medium is MS+3 mg / L 6-BA, and the preferred culture time is 5 days; in the culture medium of MS+2 mg / L 6-BA+0.2 mg / L NAA, the preferred culture time is 25-35 days;

[0034] In step S3, 0.3 mg / L 6-BA and 0.03 mg / L NAA are added to the culture medium, and the culture time is preferably 15-25 days;

[0035] In step S4, 1.0 mg / L IBA, 0.4 mg / L NAA, and 0.15 mg / L Eth are added to the culture medium, and the culture time is preferably 15-25 days;

[0036] In step S5, the culture medium is 2MS+0.8 mg / L 6-BA+0.08 mg / L NAA, and the preferred culture time is 20-30 days;

[0037] In step S6, 0.5 g / L KNO3 is added to the culture medium to promote soybean fruiting;

[0038] Artificial pollination in step S7 is used for hybridization.

[0039] Specifically, 25-35 g / L sucrose, 0.08-0.12 g / L inositol, and 4-6 g / L agar powder are added to each culture medium, and the pH value of the culture medium is adjusted to 5.5-6.5; preferably, 30 g / L sucrose, 0.1 g / L inositol, and 5 g / L agar powder are added to each culture medium, and the pH value of the culture medium is adjusted to 5.8;

[0040] The culture conditions of each step are 25±2°C, light intensity 2000-3000lx, and light 16h / d.

[0041] The present invention thus provides a method for in vitro cyclic breeding of soybeans, which comprises the following steps of:

[0042] After completing the above method, collect the mature seeds in the culture container, and repeat the above method after harvesting the seeds to achieve in vitro cycle breeding that is completely completed in the culture container.

[0043] The above-mentioned soybean in vitro cyclic breeding method itself can be used for cyclic breeding, but in order to obtain more seeds and higher breeding efficiency, it can be combined with the links in the non-culture container. Therefore, the present invention further provides a soybean in vitro living cyclic breeding method, which performs the following steps in in vitro culture:

[0044] After completing the above method, collect the mature seeds in the culture container, sow them in the seedling tray or the field, or select sterile rooted seedlings for seedling hardening and transplanting. After the seeds that have matured under natural conditions are mature, select healthy, plump and pest-free seeds for further germination, and repeat the above method to achieve in vitro living cycle breeding.

[0045] As an alternative, if a suitable season is encountered during the germination stage of soybean seeds, step S1 as described above can be replaced by sowing the seeds in a seedling tray or in the field, and after maturity, the mature seeds are harvested and the subsequent steps as described above are continued to achieve in vitro living cycle breeding.

[0046] Alternatively, if the season is suitable for soybean growth, step S6 in claim 1 or 2 is to harden and transplant the rooted seedlings, and after the seeds that have grown strong under natural conditions mature, continue the steps in the method as described above to achieve in vitro living cycle breeding of soybeans.

[0047] Specifically, in the seedling hardening and transplanting, the base cultivation matrix is ​​peat soil: vermiculite: perlite = 3:1:1, and the seeds are gradually moved to large pots until they bloom and bear fruit or transplanted to the field according to their growth status.

[0048] The beneficial effect or advantage of the present invention is that the present invention uses soybean seeds and then takes the embryo tip as the starting explant, and establishes a soybean in vitro living cycle breeding regulation method through the cycle of sterile germination, adventitious bud induction, adventitious bud elongation, adventitious bud rooting, in vitro strong seedlings, flowering induction, in vitro hybridization, fruiting regulation, seedling hardening and transplanting, in vitro fruiting, sterile germination and other steps. The method of the present invention can produce high-vigor soybean seeds under in vitro conditions, greatly shorten the soybean breeding cycle, and become a new soybean breeding approach. The approach is free from seasonal restrictions (the method of the present invention can be carried out in unsuitable seasons or regions), and all-weather breeding is achieved, even completely cycle breeding in culture bottles, and rapid and complete soybean breeding (reproduction or hybrid breeding) is achieved, providing technical support for soybean molecular breeding and seedling breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Aseptic germination of soybean seeds (5 days after soybean seed inoculation).

[0050] Figure 2 The soybean parent varieties bloom in the bottle. Parent 1: Jiaoda-18; Parent 2: Changlong No. 1.

[0051] Figure 3 Electron microscope photos of soybean pollen. A: Equatorial view of pollen under natural conditions; B: Polar view of pollen under natural conditions; C: Ornaments of pollen under natural conditions; D: Equatorial view of pollen induced by in vitro culture; E: Polar view of pollen cultured in vitro; F: Ornaments of pollen cultured in vitro.

[0052] Figure 4 Hybrid soybean flowers.

[0053] Figure 5 Soybean in vitro fruiting. A is 27 days after hybridization; B is 38 days after hybridization; C is 50 days after hybridization.

[0054] Figure 6Soybean seeds. A are naturally fruiting seeds; B are in vitro fruiting seeds.

[0055] Figure 7 Soybean in vitro living cycle breeding control system. A soybean seeds; B soybean seeds sterile germination; CF soybean embryo tip regeneration; G in vitro strong seedlings; H in vitro flowering induction; I in vitro hybridization; J in vitro fruiting; K F1 generation seeds in vitro sowing; L soybean rooted seedlings transplanting; M soybean natural fruiting. DETAILED DESCRIPTION

[0056] The specific implementation of the present invention is described in detail below, but it does not constitute a limitation of the present invention. This experiment is introduced by taking Jiaoda-18 as the hybrid female parent.

[0057] Embodiment 1

[0058] 1. Aseptic germination of parent seeds

[0059] Take full and healthy Jiaoda-18 and Tianlong No. 1 soybean seeds and soak them in clean water for 5 hours, place them on a clean bench, treat them with 75% ethanol for 30 seconds, soak them in 20% sodium hypochlorite solution for 12-20 minutes, wash them with sterile water 3-5 times, inoculate them on MS culture medium, and count the contamination and growth status of each treated material. The disinfection results of Jiaoda-18 seeds are shown in Table 1. As the sodium hypochlorite treatment time increases, the cleanliness rate gradually increases. The cleanliness rate of the sodium hypochlorite treatment for 20 minutes reaches 94%, and the survival rate gradually decreases from 100% to 91.2%. Too long a sodium hypochlorite treatment will cause some Jiaoda-18 seeds to inactivate or grow passivated. The best disinfection treatment is sodium hypochlorite for 17 minutes, with a cleanliness rate of 89.33% and a survival rate of 100%. The disinfected soybean seeds began to germinate on the 5th day ( Figure 1 ).

[0060] Table 1. Effect of disinfection methods on the sterilization effect of Jiaoda-18 seeds

[0061]

[0062] 2. Adventitious bud induction

[0063] The soybean embryo tip of step 1 was transferred to MS+3mg / L 6-BA medium and cultured for 5 days, then transferred to MS+1-3mg / L6-BA+0.1-0.3mg / L NAA to induce adventitious buds, cultured for four weeks, and the adventitious bud induction rate and the average number of adventitious buds induced were counted. The results are shown in Table 2. The addition of 2mg / L 6-BA and 0.2mg / L NAA had the best effect on inducing adventitious buds in soybean embryo tips, with an adventitious bud induction rate of 89.12% and an average number of 5.04 adventitious buds.

[0064] Table 2 Effects of 6-BA and NAA on the induction of adventitious buds at the soybean embryo tip

[0065]

[0066] 3. Elongation of adventitious buds

[0067] The adventitious buds induced in step 2 were inoculated in an adventitious bud elongation induction medium, the basic medium was MS medium, 0.1-0.5 mg / L 6-BA and 0.01-0.05 mg / L NAA were added, and cultured for three weeks, and the adventitious bud elongation rate was counted and the average adventitious bud height was calculated. The results showed (Table 3) that when the 6-BA concentration was 0.3 mg / L and the NAA concentration was 0.03 mg / L, the elongation effect of the adventitious buds was the best, the adventitious bud elongation rate was 72.83%, and the average seedling height was 5.13 cm.

[0068] Table 3 Effects of 6-BA and NAA on elongation of soybean embryo tip adventitious buds

[0069]

[0070] 4. Adventitious bud rooting

[0071] The seedlings in step 3 were transferred to 1 / 2MS+0.5-1.5mg / L IBA+0.1-0.6mg / L NAA+0.05-0.2mg / L Eth medium for rooting induction, cultured for three weeks, and the rooting rate and number of roots were counted. The results are shown in Table 4, among which 1 / 2MS+1.0mg / L IBA+0.4mg / L NAA+0.15mg / L Eth had the best effect of inducing soybean rooting, with a rooting rate of 100%, an average root number of 5.36, and the plants were well rooted with a developed root system.

[0072] Table 4. Effects of plant growth regulators on rooting of soybean adventitious buds

[0073]

[0074] 5. In vitro seedlings

[0075] The rooted seedlings in step 4 were transferred to MS+0.1-1mg / L 6-BA+0.01-0.1mg / L NAA and 2MS+0.1-1mg / L 6-BA+0.01-0.1mg / L NAA medium and cultured for 4 weeks. Statistical results show (Table 5) that with the increase of 6-BA and NAA concentrations, the soybean biomass showed a trend of first rising and then falling. Among them, when the growth regulator combination was 0.8mg / L6-BA+0.08mg / L NAA, the soybean biomass was the highest. The basic medium is best with 2MS with 2 times of macroelements added, and in this medium, the biomass of soybean is always higher than that of MS medium. Therefore, the best medium for soybean in vitro seedlings is 2MS+0.8mg / L6-BA+0.08mg / L NAA, and the soybean biomass is up to 7.27g.

[0076] Table 5. Effects of basic culture medium and plant growth regulators on soybean seedling growth

[0077]

[0078] 6. In vitro Flowering Induction

[0079] When the plant height in step 5 is equivalent to the culture container, 0.5-1.25g / L KH2PO4 is added to the culture medium and cultured for 4 weeks. Adding 1.0g / L KH2PO4 has the best induction effect. Each treatment is inoculated with 20 bottles, 1 plant is inoculated in each bottle, and repeated 3 times. The number of flower buds and the budding rate are counted. The results show (Table 6) that adding an appropriate amount of KH2PO4 to the culture medium is conducive to soybean budding and flowering. As the treatment concentration increases, the number of flower buds and the budding rate first increase and then decrease. Adding 1.0g / LKH2PO4 has the best induction effect, and the budding rate is 74.64%. The I2-KI staining method is used to detect the vitality of soybean pollen (CK) formed under natural conditions and soybean pollen induced in the bottle. The vitality of soybean pollen induced in the bottle is 92.75%, and the vitality of soybean pollen produced under natural conditions is 93.43%. Electron microscopy is used to observe and record the pollen shape, equatorial view, polar view, and ornamentation of pollen formed under natural conditions and induced in the bottle. The results showed that there was no significant difference in the vitality of pollen prepared in vitro and that formed under natural conditions (Table 6). Electron microscopy results also showed that the pollen induced by tissue culture was no different from the fertile pollen formed naturally in terms of size, shape, and ornamentation ( Figure 3 ).

[0080] Table 6. Effect of KH2PO4 on soybean flowering

[0081]

[0082] 7. Artificial pollination (in vitro hybridization)

[0083] In the clean bench, pollen from sterile soybean seedling Tianlong No. 1 (male parent) was used to artificially inseminate the pistils of unopened flowers of sterile soybean seedling Jiaoda-18, and other unopened buds were removed except for the fertilized flowers, and the artificially inseminated soybean plants were marked accordingly. Figure 4 shown.

[0084] 8. Fruiting Control

[0085] When the hybrid soybean flowers formed pods about 2-3 mm long, 0.1-1.0 g / L KNO3 was added to the culture medium, and the culture medium without KNO3 was used as a control to observe the fruit setting of soybeans and count the fruit setting rate and number of soybeans. The results are shown in Table 7. With the increase of KNO3 concentration, the fruit setting rate and number of soybeans showed a trend of first increasing and then decreasing. Among them, the effect of adding 0.5 g / L KNO3 to promote soybean fruit setting was the best, with a fruit setting rate of 83.81% and a pod number of 6.62. After the seeds matured, there was no obvious difference in size from soybeans that were fruited under natural conditions ( Figure 6 ).

[0086] Table 7. Effect of KNO3 on the solidification of soybeans in bottles

[0087]

[0088] 9. Cycle Breeding

[0089] Collect the mature seeds in the bottle in step 8 above, select healthy and plump seeds and sow them in the seedling tray in spring, the germination rate is 100%; select the rooted seedlings of soybean hybrids with strong growth and well-developed root system for seedling hardening and transplanting, the cultivation medium is peat soil: vermiculite: perlite = 3:1:1, and the transplant survival rate is counted after two weeks, and the survival rate is 100%. These transplanted soybean tissue culture seedlings are gradually moved to large pots according to their growth status until they bloom and bear fruit. After the seeds that have been solid under natural conditions mature, select healthy, plump seeds without diseases and insect pests to repeat steps 1 to 8, thereby forming a cycle of soybean in vitro breeding ( Figure 7). When each link of the cycle is under the optimal culture conditions, one soybean seed can reproduce an average of 4 sprouts that can stretch and take root, and an average of 3 seedlings among the rooted seedlings have buds and flowers, and each seedling has an average of about 11 flowers, with an average fruit set rate of 80%, and a seed maturity rate of 100%. The whole process from aseptic sowing of parental seeds to harvesting mature seeds of F1 takes a total of 151 days; if the aseptic sprouts after hybridization are transplanted to in vitro culture, the survival rate can reach 100%, and the transplanted tissue culture seedlings begin to pod on the 18th day, and the seeds begin to mature on the 75th day after transplanting. An average of 20 pods can be harvested from one seedling, and an average of 35 healthy, plump and disease-free seeds can be obtained. It takes about 180 days from aseptic sowing to harvesting a large amount of soybean seeds in vitro.

Claims

1. A method for in vitro fruiting of soybeans, characterized in that: The following steps are carried out in a culture container such as a culture bottle: S1. Aseptic germination of parent soybean seeds: Select full, disease-free and insect-free soybean seeds, sterilize them, and then inoculate them into MS medium until seedlings grow; S2 Adventitious bud induction: The embryo tips of the soybean germinated in step S1 are transferred to the first culture medium, i.e., MS+1-5 mg / L 6-BA culture medium, and cultured for 3-10 days; and then transferred to the second culture medium, i.e., MS+1-3 mg / L 6-BA+ 0.1-0.3 mg / L NAA, to induce adventitious buds; S3 adventitious bud elongation: the adventitious buds induced in step S2 are inoculated in an adventitious bud elongation induction medium and cultured until sprouts are produced, wherein the adventitious bud elongation induction medium is a basic medium of MS medium supplemented with 0.1-0.5 mg / L 6-BA and 0.01-0.05 mg / L NAA; S4 adventitious bud rooting: transfer the seedlings in step S3 to 1 / 2MS + 0.5-1.5 mg / L IBA + 0.1-0.6 mg / L NAA + 0.05-0.2 mg / L ethephon medium for rooting induction; S5 in vitro seedling cultivation: the seedlings from step S4 are transferred to a medium containing MS+0.1-1 mg / L 6-BA+0.01-0.1 mg / L NAA or 2MS+0.1-1 mg / L 6-BA+0.01-0.1 mg / L NAA for seedling cultivation; S6 In vitro flowering induction: When the seedlings in step S5 grow to the same height as the culture container, 0.4-1.25 g / L KH2PO4 is added to the culture medium and cultured until buds appear; S7 Artificial pollination: In a clean environment, take soybean pollen to artificially pollinate the pistils of unopened flowers in the culture container, remove the unopened buds other than the pollinated flowers, mark the artificially pollinated soybean plants accordingly, and continue to culture; S8 Fruiting regulation: After the soybeans in step S7 have formed pods, 0.1-1.0 g / L KNO3 is added to the culture medium until mature seeds are obtained.

2. The soybean in vitro fruiting method according to claim 1, characterized in that: In step S1, full and healthy soybean seeds are soaked in clean water for 3-6 hours, then treated with ethanol, soaked in sodium hypochlorite solution, and then washed with sterile water; preferably, after soaking in clean water for 4-6 hours, they are placed in 75% ethanol for 25-35 seconds, soaked in 20% sodium hypochlorite solution for 12-20 minutes, and washed with sterile water 3-5 times.

3. The soybean in vitro fruiting method according to claim 1, characterized in that: In step S2, the first culture medium is MS+2.8-3.2 mg / L 6-BA, and the preferred culture time is 5 days; the second culture medium is MS+1.8-2.2 mg / L 6-BA+0.18-0.22 mg / L NAA, and the preferred culture time is 25-35 days; In step S3, 0.28-0.32 mg / L 6-BA and 0.02-0.04 mg / L NAA are added to the culture medium, and the culture time is preferably 15-25 days.

4. The soybean in vitro fruiting method according to claim 1, characterized in that: In step S4, 0.8-1.2 mg / L IBA, 0.3-0.5 mg / L NAA, and 0.12-0.18 mg / LEth are added to the culture medium, and the culture time is preferably 15-25 days; In step S5, the culture medium is 2MS+0.6-1.0 mg / L 6-BA+0.06-0.10 mg / L NAA, and the preferred culture time is 20-30 days.

5. The soybean in vitro fruiting method according to claim 1, characterized in that: In step S6, 0.45-0.55 g / L KNO3 is added to the culture medium to promote soybean fruiting; Artificial pollination in step S7 is used for hybridization.

6. The soybean in vitro fruiting method according to claim 1, characterized in that: Sucrose 25-35 g / L, inositol 0.08-0.12 g / L, agar powder 4-6 g / L are added to each culture medium, and the pH value of the culture medium is adjusted to 5.5-6.5; preferably, sucrose 30 g / L, inositol 0.1 g / L, agar powder 5 g / L are added to each culture medium, and the pH value of the culture medium is adjusted to 5.8; The culture conditions for each step were 25±2°C, light intensity 2000-3000 lx, and light intensity 16 h / d.

7. A method for in vitro breeding of soybean, characterized in that: The following steps are performed in vitro: After completing the method as described in any one of claims 1 to 6, collect the strong mature seeds in the culture container, and after harvesting the seeds, continue to repeat the method as described in any one of claims 1 to 6 to achieve in vitro cycle breeding completed entirely in the culture container.

8. A method for in vitro living cycle breeding of soybean, characterized in that: The following steps are performed in vitro: After completing the method as described in any one of claims 1 to 6, collect the mature seeds in the culture container and sow them into the culture container (such as a culture bottle), a seedling tray or the ground, or select sterile rooted seedlings for seedling hardening and transplanting. After the seeds that have matured under natural conditions are mature, select healthy, plump and pest-free seeds for further germination, and repeat the method as described in any one of claims 1 to 6 to achieve in vitro living cycle breeding.

9. The soybean in vitro living cycle breeding method according to claim 8, characterized in that: If a suitable season is encountered during the seed germination stage, step S1 described in any one of claims 1 to 6 is replaced by sowing the seeds in a seedling tray or in the field, and after maturity, the mature seeds are harvested and the subsequent steps described in any one of claims 1 to 6 are continued to achieve in vitro living cycle breeding; If the season is suitable for soybean growth, step S6 in any one of claims 1 to 6 is to harden and transplant the rooted seedlings, and after the seeds mature under natural conditions, continue with the steps in any one of claims 1 to 2 to achieve in vitro living cycle breeding of soybeans.

10. The method according to claim 9, characterized in that: In the seedling hardening and transplanting, the base cultivation matrix is ​​peat soil: vermiculite: perlite = 3:1:1, and the seeds are gradually moved to large pots until they bloom and bear fruit or transplanted to the field according to their growth status.

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