CN113804632A - Identification method for genotype of cotton in efficient potassium utilization - Google Patents
Identification method for genotype of cotton in efficient potassium utilization Download PDFInfo
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- G—PHYSICS
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Abstract
The invention discloses an identification method of a genotype for efficiently utilizing potassium in cotton, belonging to the technical field of biology. The invention discloses an identification method for a genotype of potassium-efficient utilization of cotton, which comprises a water culture identification method and a paper winding identification method. The method disclosed by the invention can be used for efficiently and accurately identifying the potassium high-efficiency capability of cotton, clearly, quickly and accurately reflecting the efficient absorption and utilization of potassium fertilizer, and has important significance for creating new germplasm with high potassium utilization efficiency and solving cotton premature senility caused by potassium deficiency.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an identification method of a genotype for efficient potassium utilization of cotton.
Background
Potassium is one of three essential mineral elements for plant growth, accounts for 50% of plant ash weight, is a guarantee substance for agricultural production, and is called as a quality and stress tolerance element. Cotton is an important economic crop in China and an important strategic material for people who are related to the national planning, and the root, the stem, the leaf, the seed and the cotton fiber of the cotton have use values, wherein the use value of the cotton fiber is the largest. Compared with grain crops such as wheat and corn, cotton is a potassium-loving crop and is more sensitive to potassium. Preventing premature senility and ensuring the yield and quality of cotton fibers are important requirements for the development of the cotton industry. However, the phenomenon of potassium deficiency of soil in cotton areas in China is common, and cotton production is severely restricted. Premature senility of cotton due to potassium deficiency is a common phenomenon in production, generally causes yield reduction of about 10 percent and seriously reaches about 20 percent, and can negatively influence fiber quality.
The high efficiency of plant potassium can be divided into absorption and utilization. To ensure efficient absorption of potassium, there are two mechanisms: firstly, the plant has strong root system, and secondly, the plant has higher physiological absorption capacity. The root system is the most important organ for absorbing soil moisture and nutrients, and is also the most sensitive functional organ to moisture and nutrient changes. The growth condition of the root system is closely related to the physiological metabolism of the overground part, dry matter accumulation, cotton plant senescence and yield. In addition, the root system is closely related to biological stress resistance and abiotic stress resistance. The physiological absorption capacity can be VmaxAnd KmTo measure, VmaxDenotes the maximum rate of potassium uptake by the root system, K, in the case of a sufficient potassium supplymIt represents the concentration of the external potassium supply when the rate of potassium absorption by plants is half of the maximum absorption efficiency, and it represents the potassium absorption capacity of plants in the case where the potassium supply is limited. Previous research results show that the high potassium absorption of cotton is mainly dependent on obvious morphological potassium absorption advantages, but not physiological absorption. The in vivo utilization efficiency of potassium is closely related to biomass. Different crop varieties have great difference in potassium absorption capacity and in vivo utilization efficiency, and a method for quickly and accurately screening potassium-efficient cotton genotypes is not clear.
Therefore, the problem to be solved by the technical personnel in the field is to provide an identification method for the potassium efficient utilization genotype of cotton.
Disclosure of Invention
In view of the above, the invention provides a method for identifying the genotype of potassium-efficient utilization of cotton.
In order to achieve the purpose, the invention adopts the following technical scheme:
an identification method for the genotype of cotton potassium efficient utilization comprises a water culture identification method and a paper winding identification method;
the water culture identification method comprises the following steps:
(1) culturing the cotton seedlings with the cotyledons expanded in a potassium-adapted nutrient solution and a potassium-deficient nutrient solution respectively until the seedling reaches the 5-leaf stage (about 21 days); the culture conditions are as follows: the daytime temperature is 30 +/-2 ℃, the time is 14 hours, and the illumination intensity is 450 +/-30 mu mol m-2s-1The temperature at night is 25 +/-2 ℃ and the time is 10 hours;
(2) sampling at the 5-leaf stage of the seedling, dividing the sample into a root system and an overground part from a root-stem junction after sampling, and drying and weighing; calculating potassium efficiency value 1 ═ top biomass × 100% under the potassium deficiency condition and top biomass/potassium adaptation condition and potassium efficiency value 2 ═ root biomass × 100% under the potassium deficiency condition and root biomass/potassium adaptation condition; when the potassium efficiency value is more than or equal to 30 percent and the potassium efficiency value is more than or equal to 15 percent, defining the gene type as the high-efficiency utilization of potassium;
the roll paper identification method comprises the following steps:
1) taking different cotton genotypes as materials, uniformly placing the exposed white seeds in the middle of 2 pieces of sterile absorbent paper (about 5cm away from the top) soaked with the improved Hoagland's nutrient solution, wrapping the absorbent paper wrapped with the seeds around a sterile PVC pipe, vertically placing the sterilized absorbent paper into a plastic container containing the improved Hoagland's nutrient solution, covering the surface with a breathable plastic film, and culturing for 5 days under a cold light source until the cotton emerges and cotyledons turn green from yellow; the culture conditions are as follows: the daytime temperature is 30 +/-2 ℃, the time is 14 hours, and the illumination is 7.5 mu mol m-2s-1The night temperature is 25 +/-2 ℃, and the time is 10 hours;
2) then regulating to be strong light for 7 days, and counting root form indexes; the growth conditions are as follows: the daytime temperature is 30 +/-2 ℃, the time is 14 hours, and the illumination is 300 mu mol m-2s-1The temperature at night is 25 +/-2 ℃ and the time is 10 hours;
3) the cotton material with root biomass, the daily increase of the main root length and the side root number respectively more than 0.58 mg/day, 1.07 cm/day and 3.17/day is a potassium efficient utilization genotype.
Further, the potassium-deficient nutrient solution in the step (1) comprises the following components: ca (NO)3)22.5mmol/L，MgSO41mmol/L，NH4H2PO40.5mmol/L，KCl 0.02mmol/L，EDTA-FeNa 0.1mmol/L，CuSO42×10-4mmol/L，ZnSO41×10-3mmol/L，H3BO32×10-2mmol/L，(NH4)6Mo7O245×10-6mmol/L，MnSO41×10-3mmol/L。
Further, the potassium-adapted nutrient solution in the step (1) and the improved Hoagland nutrient solution in the step 1) have the following compositions: ca (NO)3)22.5mmol/L，MgSO41mmol/L，NH4H2PO40.5mmol/L，KCl 0.5mmol/L，EDTA-FeNa 0.1mmol/L，CuSO42×10-4mmol/L，ZnSO41×10-3mmol/L，H3BO32×10-2mmol/L，(NH4)6Mo7O245×10-6mmol/L，MnSO41×10-3mmol/L。
Water culture identification method: the biomass is a direct reflection of the high and low absorption and utilization efficiency of potassium.
And (3) identifying the roll paper: although the available potassium in the soil is mainly distributed in the soil layer of 0-40 cm, the potassium is more favorably absorbed when the root systems are distributed in multiple deep layers. Cotton is a deep-rooted plant with a straight root system, and consists of a main root deeply buried in the soil, side roots widely distributed and numerous root hairs to form a developed root system network. Therefore, the root system biomass weight, the main root length and the side root number which can be quickly and conveniently obtained can be used as judgment indexes for quickly and accurately screening the potassium-efficient cotton genotype.
According to the technical scheme, compared with the prior art, the invention discloses the identification method for the high-efficiency utilization genotype of the potassium in the cotton, which is a method capable of efficiently and accurately identifying the high-efficiency capacity of the potassium in the cotton and definitely, quickly and accurately reflecting the high-efficiency absorption and utilization of potassium fertilizer, and has important significance for creating new germplasm with high utilization efficiency of the potassium and solving cotton premature senility caused by potassium deficiency.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The nutrient solution for potassium deficiency (containing 0.02mmol/L potassium ions) has the following composition: ca (NO)3)22.5mmol/L，MgSO41mmol/L，NH4H2PO40.5mmol/L，KCl 0.02mmol/L，EDTA-FeNa 0.1mmol/L，CuSO42×10-4mmol/L，ZnSO41×10-3mmol/L，H3BO32×10-2mmol/L，(NH4)6Mo7O245×10-6mmol/L，MnSO41×10-3mmol/L。
The composition of the potassium (containing 0.5mmol/L potassium ion) nutrient solution and the modified Hoagland's nutrient solution is as follows: ca (NO)3)22.5mmol/L，MgSO41mmol/L，NH4H2PO40.5mmol/L，KCl 0.5mmol/L，EDTA-FeNa 0.1mmol/L，CuSO42×10-4mmol/L，ZnSO41×10-3mmol/L，H3BO32×10-2mmol/L，(NH4)6Mo7O245×10-6mmol/L，MnSO41×10-3mmol/L。
Example 1
A method for identifying the high-efficiency utilization genotype of potassium in cotton by using a hydroponic method comprises the following specific steps:
(1) germination: the method comprises the steps of taking a potassium low-efficiency insect-resistant cotton variety DP99B, a medium cotton seed 41 and a potassium high-efficiency non-insect-resistant cotton variety medium cotton seed 35 and a medium cotton seed 36 which are mainly planted in the production process as materials, disinfecting seeds for 30min by using 10% hydrogen peroxide, placing the seeds on a plastic container which is 25cm, 20cm, 15cm and fully contains water to wet sand, covering the seeds with yellow cloth, covering the yellow cloth with 2-3cm of sand, lifting the yellow cloth and the sand which covers the upper layer after seedlings push the yellow cloth out of the soil surface, and slowly injecting water into the plastic container to separate the seedlings from the sand for seedling transplanting. The dry weight of the seedlings and the potassium content before the seedlings are transplanted into the nutrient solution reflect the growth rate of each variety and the potassium content of the seeds. As can be seen from table 1, after germination for 7d, there was no significant difference between the non-insect-resistant cotton and the insect-resistant cotton in both dry weight and potassium content before the seedlings were transferred to the nutrient solutions of different potassium concentrations.
TABLE 1 Total dry matter weight and Total Potassium content of different varieties of cotton after germination for 7d
(2) Transplanting: 16 seedlings with consistent growth vigor are selected from each variety, 8 seedlings are transplanted into nutrient solution containing 0.02mmol/L potassium ions and 0.5mmol/L potassium ions respectively for culture. Temperature: 30 +/-2 ℃ in the daytime and 25 +/-2 ℃ at night; light source: a biological sodium lamp; illumination: the light is 14h and the dark is 10h every day; the illumination intensity is as follows: 450 +/-30 mu mol m-2s-1. 21d cotton seedlings (five-leaf stage) were planted in hydroponic conditions, and 8 plants were taken for determination of biomass and potassium content at different potassium concentrations for each variety.
(3) Measurement of biomass and potassium content: cotton seedlings were divided into aerial parts and root systems, dried at 80 ℃ for 48h, then weighed and the biomass recorded (Table 2).
TABLE 2 Effect of different Potassium concentrations on Biomass in different parts of Cotton
Note: different lower case letters after the same column value indicate significant difference at P <0.05 level, and different upper case letters after the same row value indicate significant difference at P <0.05 level.
Grinding the dried sample, sieving with 0.5mm sieve, soaking in neutral 1mol/L ammonium acetate solution for 5 hr, shaking for 30min, and filtering. The extract was subjected to atomic absorption spectrometry to determine the potassium content (Table 3). As can be seen from Table 1, there was no significant difference in the total potassium content of 4 cotton varieties after germination for 7 d. However, after 21 days of planting under the water culture condition, the potassium content of the root system and the overground part of the non-insect-resistant cotton treated by 0.02mM potassium ions is obviously higher than that of the root system and the overground part of the insect-resistant cotton (Table 3). Thus, non-pest-resistant cotton varieties are more sensitive to potassium than pest-resistant cotton varieties under low potassium conditions. In this example, the potassium efficiency values 1 and 2 of the non-insect-resistant cotton varieties were both greater than 30% and 15%, and therefore, reasonable thresholds of the potassium efficiency values 1 and 2 of the potassium high-efficiency utilization genes were proposed, that is, when the potassium efficiency value 1 was not less than 30% and the potassium efficiency value 2 was not less than 15%, the potassium high-efficiency utilization genotype was defined.
TABLE 3 Effect of different Potassium concentrations on Potassium content in different parts of Cotton
Note: different lower case letters after the same column value indicate significant difference at P <0.05 level, and different upper case letters after the same row value indicate significant difference at P <0.05 level.
(4) And (3) data analysis: analyzing the influence of different potassium concentrations on the potassium content and biomass in cotton plants, and providing a rapid identification method for the potassium efficient utilization genotype under the water culture method according to the potassium content and the biomass.
Example 2
A method for identifying the high-efficiency utilization genotype of potassium in cotton by using a paper rolling method comprises the following specific steps:
(1) seedling culture: 74 parts of materials (the number of the materials is 1-70 and the number of the materials is 81-84) introduced from a cotton germplasm resource library of the national Cotton institute of Chinese academy of agricultural sciences,The cotton research institute of the Henan institute of technology provides 10 parts of the material (No. 71-80 in Table 4), and a total of 84 parts of the cotton material is the test object. Taking 30 plump photoperiods from each genotype, sterilizing for 30min by using 25ml of 10% hydrogen peroxide, washing with tap water, and soaking in sterile water for 12h to accelerate germination; then 6 exposed seeds are selected from each genotype and evenly placed among 2 pieces of sterile absorbent paper soaked with the improved Hoagland's nutrient solution (the distance from the top is about 5cm), then the absorbent paper wrapped with the seeds is rolled around a sterile PVC pipe and vertically placed into a plastic container filled with 5L of the improved Hoagland's nutrient solution, and the surface is covered with a breathable plastic film to prevent the moisture from evaporating too fast. Firstly, under a cold light source (the daytime temperature is 30 +/-2 ℃, the time is 14h, and the illumination is 7.5 mu mol m-2s-1The night temperature is 25 +/-2 ℃, the time is 10 hours, and the illumination is 0 mu mol m-2s-1) Culturing for 5d until the cotton emerges and the cotyledon turns green from yellow; then adjusting to strong light (day temperature is 30 + -2 deg.C, duration is 14h, illumination is 300 μmol m-2s-1The night temperature is 25 +/-2 ℃, the time is 10 hours, and the illumination is 0 mu mol m- 2s-1) And culturing for 7 days until the main root and the lateral root of the cotton are completely developed. Spraying carbendazim on the surface of the paper cylinder every day to prevent the seeds from mildewing. The 84 cotton genotypes tested are shown in table 4.
TABLE 4 different Cotton genotype Numbers
(2) Index measurement
Root system phenotypic character: the main root length was measured manually and the number of macroscopic lateral roots longer than 1mm (first order lateral roots) on the main root was recorded (table 5).
Biomass: placing the overground part of the cotton seedling and the root system with the recorded main root length and side root number into a 105 ℃ oven, deactivating enzymes for 30min, then drying to constant weight at 80 ℃, weighing the overground part and the root system with a ten-thousandth balance after the sample is cooled to room temperature, and recording the biomass of the overground part and the root system (Table 5).
TABLE 5 genotype test indices
(3) Data analysis
Data analysis was performed using SPSS 22.0 software. The results show that the biomass of the root system, the length of the main root and the number of lateral roots are obviously and positively correlated with the biomass of the overground part. Therefore, clustering analysis is respectively carried out according to three indexes of root biomass, main root length and lateral root number (tables 6-8); from tables 6 to 8, it can be seen that there are 13 varieties with large biomass of root system, long main root and large number of lateral roots. The daily increase amounts of root biomass, main root length and lateral root number of 13 varieties are respectively more than 0.58 mg/day, 1.07 cm/day and 3.17/day (Table 9), so that the method for quickly identifying the potassium efficient utilization genotype under the paper rolling method is provided: the cotton material with root biomass, main root length and side root number respectively more than 0.58 mg/day, 1.07 cm/day and 3.17/day is a potassium efficient utilization genotype.
TABLE 6 clustering analysis results according to root dry weight
Note: the type I is a variety with a large root dry weight, and the type II is a variety with a small root dry weight.
TABLE 7 clustering analysis results according to principal root Length
Note: the type I is a variety with long main root, and the type II is a variety with short main root.
TABLE 8 clustering analysis results according to lateral root number
Note: the type I is a variety with more lateral roots, and the type II is a variety with less lateral roots.
TABLE 9 Potassium high Performance genotypes
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (3)
1. An identification method for the genotype of cotton with high potassium utilization efficiency is characterized by comprising a water culture identification method and a paper winding identification method;
the water culture identification method comprises the following steps:
(1) culturing the cotton seedlings with the cotyledons expanded in a potassium-adapted nutrient solution and a potassium-deficient nutrient solution respectively until the seedling reaches the 5-leaf stage; the culture conditions are as follows: the daytime temperature is 30 +/-2 ℃, the time is 14 hours, and the illumination intensity is 450 +/-30 mu molm-2s-1The temperature at night is 25 +/-2 ℃ and the time is 10 hours;
(2) sampling at the 5-leaf stage of the seedling, dividing the sample into a root system and an overground part from a root-stem junction after sampling, and drying and weighing; calculating potassium efficiency value 1 ═ top biomass × 100% under the potassium deficiency condition and top biomass/potassium adaptation condition and potassium efficiency value 2 ═ root biomass × 100% under the potassium deficiency condition and root biomass/potassium adaptation condition; when the potassium efficiency value is more than or equal to 30 percent and the potassium efficiency value is more than or equal to 15 percent, defining the gene type as the high-efficiency utilization of potassium;
the roll paper identification method comprises the following steps:
1) taking different cotton genotypes as materials, uniformly placing the exposed white seeds in the middle of 2 pieces of sterile absorbent paper soaked with improved Hoagland's nutrient solution, wrapping the absorbent paper wrapped with the seeds around a sterile PVC pipe, vertically placing the sterilized absorbent paper in a plastic container containing the improved Hoagland's nutrient solution, covering the surface with a breathable plastic film, and culturing for 5 days under a cold light source until the cotton seedlings emerge and cotyledons turn green from yellow; the culture conditions are as follows: the daytime temperature is 30 +/-2 ℃, the time is 14 hours, and the illumination is 7.5 mu mol-2s-1The night temperature is 25 +/-2 ℃, and the time is 10 hours;
2) then regulating to be strong light for 7 days, and counting root form indexes; the growth conditions are as follows: the daytime temperature is 30 +/-2 ℃, the time is 14 hours, and the illumination is 300 mu mol-2s-1The temperature at night is 25 +/-2 ℃ and the time is 10 hours;
3) the cotton material with root biomass, the daily increase of the main root length and the side root number respectively more than 0.58 mg/day, 1.07 cm/day and 3.17/day is a potassium efficient utilization genotype.
2. The method for identifying the potassium-efficient-utilization genotype of cotton according to claim 1, characterized in that the potassium-deficient nutrient solution in the step (1) comprises the following components: ca (NO)3)2 2.5mmol/L，MgSO41mmol/L，NH4H2PO4 0.5mmol/L，KCl 0.02mmol/L，EDTA-FeNa 0.1mmol/L，CuSO4 2×10-4mmol/L，ZnSO4 1×10-3mmol/L，H3BO32×10-2mmol/L，(NH4)6Mo7O24 5×10-6mmol/L，MnSO4 1×10-3mmol/L。
3. The method for identifying the potassium-efficient genotype of cotton according to claim 1, wherein the potassium-adapted nutrient solution of step (1) and the modified Hoagland nutrient solution of step 1) are composed of the following components: ca (NO)3)2 2.5mmol/L，MgSO4 1mmol/L，NH4H2PO4 0.5mmol/L，KCl 0.5mmol/L，EDTA-FeNa 0.1mmol/L，CuSO4 2×10-4mmol/L，ZnSO4 1×10-3mmol/L，H3BO3 2×10-2mmol/L，(NH4)6Mo7O24 5×10-6mmol/L，MnSO4 1×10-3mmol/L。
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