Breeding method of sugarcane variety capable of efficiently fixing nitrogen and resisting acid and aluminum

Abstract

The invention discloses a sugarcane variety breeding method capable of efficiently fixing nitrogen and resisting acid and aluminum, and relates to the technical field of sugarcane variety breeding. The method comprises the following breeding steps: (1) carrying out acid-aluminum stress treatment on tissue culture rooting seedlings of different sugarcane varieties, screening plants with roots capable of normally growing and developing, hybridizing the screened plants, carrying out the same acid-aluminum stress treatment on the first filial generation of tissue culture rooting seedlings, and selecting acid-aluminum resistant plants; and (2) soaking the roots of the sterile tissue culture rooting seedlings of the acid-resistant aluminum plants in a nitrogen-fixing bacteria solution, cultivating, not applying a nitrogen fertilizer in the whole process, selecting the robust plants with high nitrogenase activity, hybridizing for 2-3 generations, planting hybridized offspring in a nitrogen-free environment, not applying a nitrogen fertilizer in the planting process, selecting excellent plants with high nitrogenase activity, and planting for 2-3 generations. The sugarcane variety is the sugarcane variety capable of efficiently fixing nitrogen and resisting acid and aluminum. The variety bred by the invention has strong affinity for nitrogen-fixing bacteria, and can rapidly enrich a large amount of nitrogen-fixing bacteria and form an efficient combined nitrogen fixation system.

Description

Technical Field

[0001] The invention relates to the technical field of sugarcane variety breeding, and in particular to a method for breeding a sugarcane variety with high nitrogen fixation efficiency and acid and aluminum resistance. Background Art

[0002] Guangxi accounts for more than 65% of the country's sugarcane cultivation area and sugarcane production. It is the most important sugar production area in my country. It is also one of the areas with the most serious soil acidification in my country. About 800,000 hectares of sugarcane fields in the region have acidic soils with a pH below 5.0. Aluminum toxicity is the most important abiotic stress factor that restricts crop growth in acidic soils. When the soil pH drops below 5.0, the complexed and combined aluminum in the soil will gradually dissociate, releasing a large amount of active Al 3+ The plants are poisoned, causing nutritional imbalance, root poisoning, etc., which seriously affects plant growth and development. Large amounts of biased nitrogen fertilizers are one of the main causes of soil acidification. The average annual nitrogen consumption of sugarcane fields in Guangxi is about 550,000 tons, and the nitrogen content in the harvest is only 75,000 tons. The nitrogen fertilizer utilization efficiency is less than 15%. There is a serious problem of excessive nitrogen fertilizer application exacerbating soil acidification. Therefore, increasing biological nitrogen fixation and reducing the use of nitrogen fertilizers is one of the important ways to alleviate soil acidification.

[0003] As a grass plant, sugarcane mainly fixes nitrogen by forming a combined nitrogen-fixing system with nitrogen-fixing bacteria. The combined nitrogen-fixing bacteria mainly colonize the root system, stems and leaves of sugarcane, which is a loose combination. However, the combined nitrogen-fixing efficiency of sugarcane varieties in my country is relatively low, only about 5%. Studies have shown that the formation of an efficient combined nitrogen-fixing system in sugarcane is related to the long-term non-application or low application of nitrogen fertilizers. However, due to the long-term high nitrogen stress, the number of bacteria that can survive low nitrogen in sugarcane is low, making it difficult to form an efficient combined nitrogen-fixing system. Therefore, screening, cultivating and promoting nitrogen-fixing and acid-aluminum-resistant sugarcane varieties are the most fundamental and effective measures to effectively utilize acidic soils, solve the problem of excessive nitrogen fertilizer application, and improve crop productivity. At present, there are few methods for breeding sugarcane varieties that have both nitrogen fixation and acid-aluminum resistance, and further research is needed. Summary of the invention

[0004] In view of the above shortcomings, the present invention provides a method for breeding sugarcane varieties with high nitrogen fixation efficiency and acid and aluminum resistance. The method of the present invention can obtain sugarcane varieties with strong acid and aluminum resistance and strong affinity for nitrogen-fixing bacteria, which can quickly enrich a large number of nitrogen-fixing bacteria and form an efficient combined nitrogen-fixing system. The specific technical scheme is as follows:

[0005] A method for breeding sugarcane varieties with high nitrogen fixation efficiency and acid aluminum resistance comprises the following steps:

[0006] (1) subjecting tissue culture rooted seedlings of different sugarcane varieties to acid-aluminum stress treatment, selecting plants whose roots can grow and develop normally under certain pH value and aluminum ion concentration stress, hybridizing the selected plants, and subjecting the next generation tissue culture rooted seedlings to the same acid-aluminum stress treatment, selecting plants whose roots can grow and develop normally as acid-aluminum resistant plants;

[0007] (2) The roots of the sterile tissue culture rooted seedlings of the acid-aluminum resistant plants are immersed in a nitrogen-fixing bacteria solution and then transferred to a container containing a sterilized substrate for cultivation without applying nitrogen fertilizer throughout the process. The agronomic characteristics of the sugarcane are observed during the elongation period and the nitrogenase activity is measured. The plants with high nitrogenase activity and strong stamina are selected for 2-3 generations of hybridization. The hybrid offspring are all planted in a nitrogen-free environment, and no nitrogen fertilizer is applied during the planting process. The agronomic characteristics are observed and the nitrogenase activity is measured during the elongation period. Finally, the plants with high nitrogenase activity and strong stamina are selected, which are the sugarcane varieties with high nitrogen fixation efficiency and acid-aluminum resistance.

[0008] Furthermore, in step (1), the acid-aluminum stress treatment is: 10-20 tissue culture rooted seedlings of each sugarcane variety are selected for the test, the test group and the control group are set with the same number of seedlings, the roots of the seedlings in the test group are immersed in a pH value of 3.5-4.5, Al 3+ The roots of the seedlings in the control group were immersed in a nutrient solution with a pH value of 6 in a concentration of 100-200 μmol / L. After culturing the test group and the control group at 20-28°C for 20-30 days, the average root weight and root activity of each group were measured. Compared with the control group, the test group's root weight and root activity changed within a range of less than 10%, indicating that the roots of the plants can grow and develop normally.

[0009] Furthermore, the nutrient solution is Hoagland's nutrient solution.

[0010] Furthermore, in step (2), the nitrogen-fixing bacteria solution comprises: 10-20 parts of Acetobacter diazotrophicus, 25-35 parts of Raoultella, 4-9 parts of Klebsiella and 800-1000 parts of water.

[0011] Furthermore, in step (2), the soaking time is 8-10 hours.

[0012] Furthermore, in step (2), the matrix comprises the following raw materials in parts by weight: 50-80 parts of sand, 20-45 parts of perlite, 7-16 parts of vermiculite and 7-13 parts of decomposed straw organic fertilizer.

[0013] Furthermore, in step (2), the agronomic traits refer to plant height, root weight and root length; robust refers to tall plants, many tillers and a well-developed root system.

[0014] Furthermore, in step (2), the high nitrogenase activity means that the nitrogenase activity is 400 mg / L or more.

[0015] Furthermore, during the hybridization, a stimulant is sprayed on the female parent flower spike one day before pollination; the stimulant includes 0.05-0.10 parts of cytokinin, 2-8 parts of fructose, 0.1-0.4 parts of arginine, 0.3-0.8 parts of proline and 0.01-0.03 parts of vitamin B1.

[0016] Furthermore, when spraying the stimulant, it is first diluted 800-1000 times with water and then sprayed until the flower spike is moistened.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. The present invention first performs acid-aluminum stress treatment to select plants with certain acid-aluminum resistance as hybrid parents, in order to improve the breeding accuracy, avoid blindly selecting parents, and speed up the breeding speed. Among them, acid-aluminum stress treatment can quickly lock varieties with certain acid-aluminum resistance, and use these plants as hybrid parents in the next step, so that more and more suitable target plants can appear in their offspring, thereby speeding up the breeding speed. Using plants with certain acid-aluminum resistance as hybrid parents can further obtain stable acid-aluminum resistance varieties.

[0019] 2. The present invention uses nitrogen-fixing bacteria to infect the roots of stable and acid-aluminum-resistant varieties and cultivate them without nitrogen, and selects plants with high nitrogenase activity as later hybrid parents, in order to improve the accuracy of breeding, avoid blind selection of parents, and speed up the breeding. Among them, nitrogen-fixing bacteria infect the roots and cultivate them without nitrogen, which can quickly screen out plants with more root secretions, stronger affinity for nitrogen-fixing bacteria, and the ability to quickly enrich nitrogen-fixing bacteria. These plants are used as hybrid parents in the next step, and their offspring can produce more and more suitable target plants, thereby speeding up the breeding speed.

[0020] 3. The present invention hybridizes plants with high nitrogenase activity and robustness for 2-3 generations, and the offspring of the hybridization are all planted in a nitrogen-free environment, and no nitrogen fertilizer is applied during the planting process, so that a stable high nitrogen-fixing variety can be finally obtained. Because the nitrogen-fixing ability of the endogenous nitrogen-fixing bacteria of the sugarcane plant can be continuously enhanced under the condition of long-term nitrogen deficiency, the nitrogen-fixing bacteria can stably combine with the sugarcane to fix nitrogen, so that the sugarcane is continuously trained into a high nitrogen-fixing variety.

[0021] 4. In the present invention, stimulants are sprayed on the female flower spikes during hybridization. The cytokinin, fructose, arginine, proline and vitamin B1 in the stimulants can synergistically promote the development of female flowers, increase the production of stigma mucus, and improve the adhesion of the stigma to pollen, thereby improving the pollination success rate and fruit setting rate, and accelerating the breeding speed. DETAILED DESCRIPTION

[0022] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments.

[0023] Example 1

[0024] A method for breeding sugarcane varieties with high nitrogen fixation efficiency and acid aluminum resistance comprises the following steps:

[0025] (1) subjecting tissue culture rooted seedlings of different sugarcane varieties to acid-aluminum stress treatment, selecting plants whose roots can grow and develop normally under certain pH value and aluminum ion concentration stress, hybridizing the selected plants, and subjecting the next generation tissue culture rooted seedlings to the same acid-aluminum stress treatment, selecting plants whose roots can grow and develop normally as acid-aluminum resistant plants;

[0026] The acid-aluminum stress treatment is as follows: 20 tissue culture rooted seedlings of each sugarcane variety are selected for the test, the test group and the control group are set with the same number of seedlings, the roots of the seedlings in the test group are immersed in an acid-aluminum stress solution with a pH value of 4 and an Al 3+ The roots of the seedlings in the control group were immersed in Hoagland's nutrient solution with a concentration of 200 μmol / L. After the test group and the control group were cultured at 28°C for 20 days, the average root weight and root activity of each group were measured. Compared with the control group, the test group's root weight and root activity changed within a range of less than 10%, indicating that the roots of the plants can grow and develop normally.

[0027] When weighing the root system, the plant was first taken out of the hydroponic system, the root system was separated from the plant, and the surface moisture was wiped dry with cotton cloth. The root activity was determined using the triphenyltetrazolium chloride method (TTC method).

[0028] (2) The roots of the sterile tissue culture rooted seedlings of the acid-aluminum-resistant plants are immersed in a nitrogen-fixing bacteria solution for 8 hours, and then transferred to a container containing a sterilized substrate for cultivation. No nitrogen fertilizer is applied during the entire process. During the elongation period, agronomic traits such as plant height, root weight and root length of the sugarcane are observed, and the nitrogenase activity (the amount of reduced ethylene, measured as the content of ethylene produced in the acetylene reduction reaction, the same below) is measured. Plants with a nitrogenase activity of more than 400 mg / L, tall plants, many tillers, and well-developed root systems are selected for two generations of hybridization. The hybrid offspring are all planted in a nitrogen-free environment, and no nitrogen fertilizer is applied during the planting process. The agronomic traits are observed and the nitrogenase activity is measured during the elongation period. Finally, plants with the same nitrogenase activity and agronomic traits as the above are selected, which are the sugarcane varieties with high nitrogen fixation efficiency and acid-aluminum resistance.

[0029] The nitrogen-fixing bacteria liquid includes: 10 parts of nitrogen-fixing Acetobacter diazotrophicus, 25 parts of Raoultella LO3, 4 parts of Klebsiella and 800 parts of water. The strains are obtained by separation and screening from sugarcane plants and rhizosphere soil samples by the applicant in the early stage. It should be noted that the strains of related genera purchased from the market also have the same effect. The matrix includes the following raw materials in parts by weight: 50 parts of sand, 20 parts of perlite, 7 parts of vermiculite and 7 parts of straw decomposed organic fertilizer. During the above hybridization, the female parent flower spike is sprayed with stimulants one day before pollination. When spraying, it is diluted 800 times with water and sprayed until the flower spike is wet; the stimulants include 0.05 parts of cytokinin, 2 parts of fructose, 0.1 parts of arginine, 0.3 parts of proline and 0.01 parts of vitamin B1.

[0030] Example 2

[0031] A method for breeding sugarcane varieties with high nitrogen fixation efficiency and acid aluminum resistance comprises the following steps:

[0032] (1) subjecting tissue culture rooted seedlings of different sugarcane varieties to acid-aluminum stress treatment, selecting plants whose roots can grow and develop normally under certain pH value and aluminum ion concentration stress, hybridizing the selected plants, and subjecting the next generation tissue culture rooted seedlings to the same acid-aluminum stress treatment, selecting plants whose roots can grow and develop normally as acid-aluminum resistant plants;

[0033] The acid-aluminum stress treatment is as follows: 10 tissue culture rooted seedlings of each sugarcane variety are selected for the test, the test group and the control group are set with the same number of seedlings, the roots of the seedlings in the test group are immersed in an acid-aluminum stress solution with a pH value of 3.5 and an Al 3+ The roots of the seedlings in the control group were immersed in Hoagland's nutrient solution with a concentration of 100 μmol / L. After the test group and the control group were cultured at 28°C for 20 days, the average root weight and root activity of each group were measured. Compared with the control group, the test group's root weight and root activity changed within a range of less than 10%, indicating that the roots of the plants can grow and develop normally.

[0034] When weighing the root system, the plant was first taken out of the hydroponic system, the root system was separated from the plant, and the surface moisture was wiped dry with cotton cloth. The root activity was determined using the triphenyltetrazolium chloride method (TTC method).

[0035] (2) The roots of the sterile tissue culture rooted seedlings of the acid-aluminum-resistant plants are placed in a nitrogen-fixing bacteria solution and soaked for 10 hours, and then transferred to a container containing a sterilized substrate for cultivation. No nitrogen fertilizer is applied during the entire process. During the elongation period, agronomic traits such as plant height, root weight and root length of the sugarcane are observed, and the nitrogenase activity is measured. Plants with a nitrogenase activity of more than 400 mg / L, tall plants, many tillers, and well-developed root systems are selected for three generations of hybridization. The hybrid offspring are all planted in a nitrogen-free environment, and no nitrogen fertilizer is applied during the planting process. The agronomic traits and nitrogenase activity are observed during the elongation period. Finally, plants with the same nitrogenase activity and agronomic traits as mentioned above are selected, which are the sugarcane varieties with high nitrogen fixation efficiency and acid-aluminum resistance.

[0036] The nitrogen-fixing bacteria liquid includes: 20 parts of nitrogen-fixing Acetobacter diazotrophicus, 35 parts of Raoultella LO3, 9 parts of Klebsiella and 1000 parts of water. The strains are obtained by separation and screening from sugarcane plants and rhizosphere soil samples by the applicant in the early stage. It should be noted that the strains of related genera purchased from the market also have the same effect. The matrix includes the following raw materials in parts by weight: 80 parts of sand, 45 parts of perlite, 16 parts of vermiculite and 13 parts of straw decomposed organic fertilizer. During the above hybridization, the female parent flower spike is sprayed with stimulants one day before pollination. When spraying, it is diluted 1000 times with water and sprayed until the flower spike is wet; the stimulants include 0.10 parts of cytokinin, 8 parts of fructose, 0.4 parts of arginine, 0.8 parts of proline and 0.03 parts of vitamin B1.

[0037] In 2023, the applicant planted the variety selected and bred in Example 1 in Dingdang Town, Longan County, Guangxi. The soil type in the planting area is sandy loam. The physical and chemical properties of the sugarcane soil before planting are as follows: organic matter content 20.09g / kg, total nitrogen content 0.124%, total phosphorus content 0.036%, total potassium content 0.51%, pH 4.5-4.7, acidic soil. The planting sugarcane row is 10m long, with a row spacing of 1.5m, and is planted in 7 plots, with 5 rows planted in each plot, 3 replicates, and the average value is taken when statistical data is collected. Plot 1 applied nitrogen fertilizer according to conventional methods during the planting process (conventional nitrogen application method: 20 kg / mu of nitrogen fertilizer in base fertilizer, 15 kg / mu of nitrogen fertilizer in tillering stage, 15 kg / mu of nitrogen fertilizer in jointing stage, and 15 kg / mu of nitrogen fertilizer in tail fertilizer); Plot 2 did not apply any nitrogen fertilizer during the planting process; Plot 3 applied 10% of the conventional amount of nitrogen fertilizer during the planting process; Plot 4 applied 20% of the conventional amount of nitrogen fertilizer during the planting process; Plot 5 applied 25% of the conventional amount of nitrogen fertilizer during the planting process; Plot 6 applied 30% of the conventional amount of nitrogen fertilizer during the planting process. Observe the chlorosis of the leaf tips of the plants during the elongation period. More than 80% of the leaves withered are severely chlorosis, and 10-20% of the leaves withered are mildly chlorosis (mildly chlorosis means acid and aluminum resistance, and severe chlorosis means acid and aluminum resistance). Measure the nitrogenase activity of sugarcane during the elongation period (acetylene reduction method, the amount of reduced ethylene, measured by the ethylene content produced in the acetylene reduction reaction), and measure the yield during the maturity period. The results are shown in Table 1:

[0038] Table 1 Table 1 Acid-resistant aluminum in various embodiments and comparative examples

[0039] project Nitrogenase activity (mg / L) <![CDATA[Yield (t / hm 2 )]]> Plant leaf tip Plot 1 (normal nitrogen fertilizer application) 386.2 68.6 No chlorosis Plot 2 (no nitrogen fertilizer) 425.4 63.1 No chlorosis Plot 3 (nitrogen application rate is 10% of that in Plot 1) 433.1 65.9 No chlorosis Plot 4 (nitrogen application rate is 20% of that in Plot 1) 437.4 66.5 No chlorosis Plot 5 (nitrogen application rate is 25% of that in Plot 1) 438.2 68.5 No chlorosis Plot 6 (nitrogen application rate is 30% of that in Plot 1) 435.0 68.4 No chlorosis

[0040] Table 1 shows that the sugarcane varieties bred by the method of the present invention are planted on acid-aluminum soil, and the leaf tips of the plants have no chlorosis and no poisoning during the elongation period, indicating that the sugarcane varieties bred by the method of the present invention have the characteristics of acid-aluminum resistance. From the yield data of each plot, the yields of plot 1, plot 5 and plot 6 are basically the same, indicating that when planting the sugarcane varieties bred by the method of the present invention, there is no need to apply too much nitrogen fertilizer, and only about 25% of the conventional nitrogen application amount is needed. Excessive nitrogen fertilizer application does not contribute to the increase in yield, but only leads to aggravated soil acidification, and also inhibits the nitrogenase activity of sugarcane (plot 1 has the largest nitrogen application amount but the lowest nitrogenase activity), affecting the nitrogen fixation efficiency of sugarcane.

[0041] In summary, the method of the present invention can obtain a sugarcane variety that has strong acid and aluminum resistance and strong affinity for nitrogen-fixing bacteria, can quickly enrich a large number of nitrogen-fixing bacteria and form an efficient combined nitrogen-fixing system.

[0042] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.

Claims

1. A method for breeding sugarcane varieties with high nitrogen fixation efficiency and acid and aluminum resistance, characterized in that: The following steps are involved: (1) subjecting tissue culture rooted seedlings of different sugarcane varieties to acid-aluminum stress treatment, selecting plants whose roots can grow and develop normally under certain pH value and aluminum ion concentration stress, hybridizing the selected plants, and subjecting the next generation tissue culture rooted seedlings to the same acid-aluminum stress treatment, selecting plants whose roots can grow and develop normally as acid-aluminum resistant plants; (2) The roots of the sterile tissue culture rooted seedlings of the acid-aluminum resistant plants are immersed in a nitrogen-fixing bacteria solution and then transferred to a container containing a sterilized substrate for cultivation without applying nitrogen fertilizer throughout the process. The agronomic characteristics of the sugarcane are observed during the elongation period and the nitrogenase activity is measured. The plants with high nitrogenase activity and strong stamina are selected for 2-3 generations of hybridization. The hybrid offspring are all planted in a nitrogen-free environment, and no nitrogen fertilizer is applied during the planting process. The agronomic characteristics are observed and the nitrogenase activity is measured during the elongation period. Finally, the plants with high nitrogenase activity and strong stamina are selected, which are the sugarcane varieties with high nitrogen fixation efficiency and acid-aluminum resistance.

2. The method for breeding sugarcane varieties with high nitrogen fixation efficiency and acid aluminum resistance according to claim 1, characterized in that: In step (1), the acid-aluminum stress treatment is: 10-20 tissue culture rooted seedlings of each sugarcane variety are selected for the test, the test group and the control group are set with the same number of seedlings, the roots of the seedlings in the test group are immersed in a pH value of 3.5-4.5, Al 3+ The roots of the seedlings in the control group were immersed in a nutrient solution with a pH value of 6 in a concentration of 100-200 μmol / L. After culturing the test group and the control group at 20-28°C for 20-30 days, the average root weight and root activity of each group were measured. Compared with the control group, the test group's root weight and root activity changed within a range of less than 10%, indicating that the roots of the plants can grow and develop normally.

3. The method for breeding sugarcane varieties with high nitrogen fixation efficiency and acid aluminum resistance according to claim 2, characterized in that: The nutrient solution is Hoagland's nutrient solution.

4. The method for breeding sugarcane varieties with high nitrogen fixation efficiency and acid aluminum resistance according to claim 1, characterized in that: In step (2), the nitrogen-fixing bacteria solution comprises: 10-20 parts of nitrogen-fixing Acetobacter, 25-35 parts of Raoultella, 4-9 parts of Klebsiella and 800-1000 parts of water.

5. The method for breeding sugarcane varieties with high nitrogen fixation efficiency and acid aluminum resistance according to claim 1, characterized in that: In step (2), the soaking time is 8-10 hours.

6. The method for breeding sugarcane varieties with high nitrogen fixation efficiency and acid aluminum resistance according to claim 1, characterized in that: In step (2), the matrix comprises the following raw materials in parts by weight: 50-80 parts of sand, 20-45 parts of perlite, 7-16 parts of vermiculite and 7-13 parts of decomposed straw organic fertilizer.

7. The method for breeding sugarcane varieties with high nitrogen fixation efficiency and acid aluminum resistance according to claim 1, characterized in that: In step (2), the agronomic traits refer to plant height, root weight and root length.

8. The method for breeding sugarcane varieties with high nitrogen fixation efficiency and acid aluminum resistance according to claim 1, characterized in that: In step (2), the high nitrogenase activity means that the nitrogenase activity is 400 mg / L or more.

9. The method for breeding sugarcane varieties with high nitrogen fixation efficiency and acid aluminum resistance according to claim 1, characterized in that: During the hybridization, the female parent flower spike is sprayed with stimulants one day before pollination; the stimulants include 0.05-0.10 parts of cytokinin, 2-8 parts of fructose, 0.1-0.4 parts of arginine, 0.3-0.8 parts of proline and 0.01-0.03 parts of vitamin B1.

10. The method for breeding sugarcane varieties with high nitrogen fixation efficiency and acid aluminum resistance according to claim 9, characterized in that: When spraying the stimulant, dilute it 800-1000 times with water and spray it until the flower spike is moistened.