CN106105727B - Identification method for sodium salt resistance of semi-wild cotton in germination period - Google Patents

Abstract

The invention relates to plant stress-resistant physiology, in particular to a method for identifying sodium salt resistance of semi-wild cotton in a germination period. The method comprises the following steps: 1) homogenizing semi-wild cotton seeds; 2) preparation of salt-stressed matrix: 3) sowing and cultivating cotton seedlings; 4) and (5) counting and analyzing results. The invention provides an identification method of salt resistance of semi-wild cotton in a germination period, so that a semi-wild cotton material with high salt resistance can be rapidly and accurately screened, the salt resistance genetic improvement of cultivated cotton can be accelerated, and the method has practical utilization value and significance.

Description

Identification method for sodium salt resistance of semi-wild cotton in germination period

Technical Field

The invention relates to plant stress-resistant physiology, in particular to a method for identifying sodium salt resistance of semi-wild cotton in a germination period.

Background

Semi-wild cotton (semi-wild cotton) is a wild species of gossypium hirsutum, 7 in total: broad-leaved cotton (latifolium), permel cotton (palmeri), maligater cotton (marie-galante), leiomycosis cotton (richmondi), yucatanese cotton (yucatanese), molel cotton (morrilli) and pointe cotton (punctatum). The half-wild cotton origin is mainly in Mexico and a small number in Brazil, the species division is based on the main characteristics of the geographical position and the shape which are mainly naturally distributed, and each species line comprises a plurality of variation types. The semi-wild cotton is the original type of upland cotton, is not domesticated artificially, has abundant genetic diversity due to the special geographical position and changeable climatic conditions, contains various excellent genes including disease and insect resistance, drought resistance, salt and alkali resistance, high fiber strength and the like compared with a cultivated variety subjected to artificial and natural double selection pressure, and has extremely important potential application value because the wild species and the cultivated variety of the upland cotton have genetic affinity and are easy to hybridize successfully.

At present, most of the researches on the salt resistance of cotton are concentrated on the research on the salt resistance of cultivated cotton (cultivated cotton), however, China is not the origin of cotton, and the genetic diversity analysis of cultivated cotton species in upland China finds that breeding resources are mostly from few upland cotton germplasm resource materials such as Dai cotton, and the like, so that the salt resistance screening of the population with rich genetic diversity is difficult to carry out. Meanwhile, in the research of NaCl germination period resistance of the cultivated cotton, 0.5% NaCl solution and 0.4% underground saline solution are mostly adopted in a field or are directly stressed by seawater, 150mM NaCl is suitable for salt stress concentration and 200mM NaCl is a limit concentration when the cultivated cotton in a laboratory is identified, the stress concentration is low, stress components are not exact and the like, so that the semi-wild cotton material with resistance diversity cannot be identified, and the defects of uneven salt distribution, large environmental influence, long identification period (about 45 days) and the like exist when the salt pond is identified, so that the repeatability of experimental results is poor. Therefore, a method suitable for the salt resistance of the semi-wild cotton in the germination period is explored to quickly and accurately screen out the semi-wild cotton material with high salt resistance, so that the salt resistance genetic improvement of the cultivated cotton can be accelerated, and the method has practical utilization value and significance.

Disclosure of Invention

The invention aims to provide a method for identifying the sodium salt resistance of semi-wild cotton in the germination period, which is used for making up the defects of a method for identifying the salt resistance of semi-wild cotton materials, so that the semi-wild cotton germplasm resources of cotton can be rapidly and accurately objectively identified, and a basis is provided for the genetic improvement of the salt resistance of cotton.

The identification method for the germination period sodium salt resistance of the semi-wild cotton is characterized by comprising the following steps of:

1) homogenizing semi-wild cotton seeds: the method comprises the steps of firstly, delinting selected semi-wild cotton materials by concentrated sulfuric acid, washing the materials by clear water, disinfecting the materials by sodium hypochlorite, then placing the materials in a 40 oven for heating and drying for 24 hours to break seed dormancy, and cutting off a small opening at the bead hole end of the seeds to expose kernels. Because the semi-wild cotton material is not easy to collect and preserve, aiming at the semi-wild cotton material of different years, the semi-wild cotton material seeds treated by the method can be kept in a consistent state before germination, so that the influence on the germination state due to the self specificity of certain materials is avoided, the condition such as pointy cotton 6 appears in the experiment, the germination rate is almost zero when the cutting and stripping treatment is not carried out, and the germination rate reaches more than 90 percent after the treatment;

2) preparation of salt-stressed matrix: adopting sand with the grain diameter of 0.05-0.80mm, washing with clear water, sterilizing at high temperature, and mixing the raw materials by weight of 100g of sand: adding NaCl solution with salt concentration of 250mM into 16ml of salt solution, and uniformly mixing to obtain the germination substrate. This can fix the water content of the sand bed at a constant 16%. The method selects 250mM NaCl concentration which is higher than the identification salt concentration of the cultivated cotton, is specially designed aiming at the resistance diversity of the semi-wild cotton, can accurately identify the semi-wild cotton material with high salt resistance, and finds that the semi-wild cotton material has the phenomena that a plurality of materials have extremely low salt damage rate and are very close to each other by taking 200mM salt as the stress concentration in the previous experiment;

3) sowing and cultivating cotton seedlings: adopting a 12cm by 19cm by 13cm germination box, putting the matrix obtained in the step 2) into the germination box, uniformly putting the seeds into the germination box, then covering a layer of matrix, and germinating and culturing for 14d under the conditions of 28 ℃ of temperature, 85% of humidity, 12h of illumination and 12h of darkness. The method can keep the consistency of the state of each material in the germination box, and avoids the occurrence of identification errors caused by external environment change;

4) statistics and result analysis: and when the cotton seedlings grow to 14 days, counting the germination number, investigating the salt damage condition of each cotton seedling, evaluating the salt damage index of each cotton seedling, and evaluating the resistance level of the semi-wild cotton material.

The invention relates to a method for identifying the salt resistance of semi-wild cotton in the germination period, which has the following advantages:

1. the germination state of the semi-wild cotton material is consistent: the semi-wild material after homogenization treatment can reach the consistency of the initial germination state, the cotton seedling emergence is uniform, the material and sand are subjected to early-stage sterilization treatment, the interference of bacteria is avoided, the seedling emergence of the contrast material in the experiment is uniform and uniform, the root system is completely developed, the germination period is uniform, and the identification accuracy is guaranteed.

2. The salt concentration specially selected for the semi-wild cotton material and suitable for the semi-wild cotton resistance polymorphism is 250mM, which is much higher than that of the semi-wild cotton material with resistance diversity by using 0.4% salt and 200mM NaCl in greenhouse culture identification.

3. The method is not controlled by seasons, has short identification period, and can identify the salt resistance of the semi-wild cotton material in a short time. The identification method of the salt resistance of the semi-wild cotton in the germination period has the identification period of 14d, the germination rate is counted 14d after sowing, and the growth condition of each cotton seedling is counted and scored. Compared with the field salt pond identification (about 45 days), the method shortens 30 days, can quickly screen the salt resistance of the semi-wild cotton material in the germination period, and provides technical support for accelerating the breeding of stress resistance.

4. The input manpower and material resources are small, and the labor and the economy are realized. The method for identifying the salt resistance of the semi-wild cotton in the germination period can be used for identifying in a greenhouse, and the method uses commercial river sand, analytical reagent and a seed germination box, and all uses high-temperature disinfection, so that the investment of a large amount of manpower and material resources is saved, and the identification of large-batch materials can be carried out; easy to popularize and utilize.

Detailed Description

Example 1 salt resistance levels of semi-wild cotton (broadleaf 40, broadleaf 27, spinelle 25, broadleaf 32, broadleaf 120, mariga-rand 85) and cultivated cotton (zhongmian 12, zhongmian 16, kaempferia numbers 1, sea 7124) were identified and evaluated using single salt NaCl stress treatment during germination.

1. Cotton material:

semi-wild cotton (broad leaf cotton 40, broad leaf cotton 27, sharp leaf cotton 25, broad leaf cotton 32, broad leaf cotton 120, marlgarand cotton 85), Bacjing huh-1, sea 7124, Gossypium barbadense 12 in the control material for cultivating cotton salt-sensitive, and Gossypium barbadense 16 in the control material for cultivating cotton salt-resistant.

2. Homogenizing semi-wild cotton seeds:

concentrated sulfuric acid delinting is carried out on selected semi-wild cotton materials, after cleaning and airing, seeds are disinfected by NaClO, after cleaning and airing, the seeds are placed in an oven at 40 ℃ for 24 hours to break seed dormancy, the seeds with full and mature seeds are selected, a single-side blade is used for cutting small openings with the size of 1mm multiplied by 1mm at the joint end (blunt end) of the seeds, and the seeds are cut to expose kernels.

3. Preparation of salt-stressed matrix:

selecting sand with the diameter of 0.05-0.80mm, washing with clear water until the sand is not turbid, air-drying, and sterilizing at 180 ℃ for 2 h; weighing the sand required by the test, and mixing the sand with the mass ratio of 100g of sand: calculating the required water quantity according to the proportion of 16ml of water, calculating the required salt according to the water quantity, preparing 250mM NaCl salt solution by using sterile water, adding the prepared salt solution into the weighed sand, and uniformly mixing to obtain a salt stress culture medium; adding sterile water into the reference matrix according to the mass ratio of 16% (water/dry sand), and uniformly mixing to obtain a reference culture matrix;

4. sowing and cultivating cotton seedlings:

firstly, paving sand with proper thickness on the lower layer of a germination box, wherein the sand is about 10-15mm, uniformly sowing seeds on wet sand, covering wet sand with 10-15mm, sowing 6 rows in each box, sowing 5 seeds in each row, and arranging one material (or repeating materials) in each two rows, covering, and culturing for 14d under the conditions of 28 ℃ of temperature, 85% of humidity, 12h of illumination and 12h of darkness; the control group was set in the same manner.

5. Statistics and result analysis:

the rate of emergence for each half-wild cotton material was counted and scored for each cotton seedling 14d after sowing. Objective grading scoring of each cotton seedling is the key to the experiment. And evaluating the resistance of the semi-wild cotton according to the emergence rate and the scoring result.

Injury scoring was performed according to criteria: the relative salt damage rate of the material was then calculated.

0 min-ungerminated seeds;

1 minute-plant height is below 50% of the contrast, and the seeds are just broken and are not broken;

the 2 min-plant height is 50% -70% of the control, and the edge of the cotyledon is curled;

the 3 min-plant height is 70-100% of the contrast, and the cotyledon curls and yellows;

the plant height of 4 min is equal to that of the control treatment, cotton seedlings grow normally, and cotyledons are unfolded.

The relative salt damage rate calculation formula is as follows:

the score rate is ∑ fx/(4n), f is the number of plants of the grade, x is the score of the corresponding grade, and n is the number of seeds.

The results are shown in Table 2:

TABLE 1 salt tolerance grading Standard for Germination period

Rank of	Relative salt damage ratio (%)	Salt tolerance
			1	0.0～20.0	High durability
2	20.1～40.0	Durable
			3	40.1～60.0	Middle tolerance
4	60.1～80.0	Mianmin
			5	80.1～100.0	Sensitivity of

Table 2: evaluation result of salt resistance level of semi-wild cotton

Claims (2)

1. The identification method for sodium salt resistance of semi-wild cotton in the germination stage is characterized by comprising the following steps:

1) homogenizing semi-wild cotton seeds: removing down selected semi-wild cotton material with concentrated sulfuric acid, washing with clear water, sterilizing with sodium hypochlorite, heating to dry to break seed dormancy, cutting off a small opening at the bead hole end of the seed to expose kernel;

2) preparation of salt-stressed matrix: adopting sand with the grain diameter of 0.05-0.80mm, cleaning, sterilizing at high temperature, and mixing the raw materials in a proportion of 100g sand: adding NaCl solution with the salt concentration of 250mM into 16ml of salt solution, and uniformly mixing to obtain a germination substrate;

3) sowing and cultivating cotton seedlings: uniformly placing the seeds into a germination box, and germinating and culturing for 14d under the conditions of 28 ℃ of temperature, 85% of humidity, 12h of illumination and 12h of darkness;

4) statistics and result analysis: and when the cotton seedlings grow to 14 days, counting the germination number, investigating the salt damage condition of each cotton seedling, evaluating the salt damage index of each cotton seedling, and evaluating the resistance level of the semi-wild cotton material.

2. The identification method of germination-stage sodium salt resistance of semi-wild cotton according to claim 1,

the relative salt damage rate calculation formula is as follows:

wherein the score rate is ∑ fx/(4n), f is the number of plants of the grade, x is the score of the corresponding grade, and n is the number of seeds sowed

The dividing standard of the score is as follows:

0 min-ungerminated seeds;

1 minute-plant height is below 50% of the contrast, and the seeds are just broken and are not broken;

the 2 min-plant height is 50% -70% of the control, and the edge of the cotyledon is curled;

the 3 min-plant height is 70-100% of the contrast, and the cotyledon curls and yellows;

the plant height of 4 min is equal to that of the control treatment, cotton seedlings grow normally, and cotyledons are unfolded.