LU501396B1

LU501396B1 - Method for identifying salt tolerance of cotton

Info

Publication number: LU501396B1
Application number: LU501396A
Authority: LU
Inventors: Dongmei Zhang; Jianlong Dai; Yanjun Zhang; Lin Sun; Weijiang Li; Zhenhuai Li; Shizhen Xu; Hezhong Dong
Original assignee: Shandong Acad Of Agricultural Sciences
Priority date: 2022-02-07
Filing date: 2022-02-07
Publication date: 2023-08-07

Abstract

The disclosure discloses a method for identifying salt tolerance of cotton. The method comprises the steps of, selecting robust cotton seedlings with 1 true leaf, treating with a nutrient solution containing 150 mM NaCl for 24 hours, selecting lateral roots with good growth state, shearing root tips, and pre-treating the root tips in a test buffer solution for 30 minutes; and fixing the root tips in a culture dish containing fresh test liquid, measuring by a using a non- invasive micro-test system, performing data analysis combined with Mageflux software to obtain outflow velocity of Na+ and K+ of the root tips, and calculating Na+/K+. The larger the Na+ outflow velocity, the smaller the K+ outflow velocity, or the larger the Na+/K+ flow velocity ratio, the stronger the salt tolerance. The method has the characteristics of non-invasive test, accurate data and fast identification, and can be used for identification of salt tolerance of both cotton and other plants.

Description

BL-5397

METHOD FOR IDENTIFYING SALT TOLERANCE OF COTTON LU501396

TECHNICAL FIELD

[01] The present disclosure relates to a method for identifying salt tolerance of cotton, in particular to a method for measuring Na” and K* flow velocity of a cotton root system under salt stress by using non-invasive micro-test technique so as to identify the salt tolerance of cotton.

BACKGROUND ART

[02] In 1974, the neuroscientist Jaffe of the Marine Biological Laboratory pf the U.S. first proposed the original concept of non-invasive micro-test technique. In 1990, the non-invasive micro-test technique was successfully applied to measuring the Ca?" flow velocity of cells. At present, the technique has been widely used in the fields of life science, environmental science, material science and the like. The non-invasive micro-test technique can determine information of Ca”, H*, K*, Na”, CI’ plasma flow velocity and three-dimensional motion direction, and has the characteristics of living, dynamic, real-time, internal and external measurement, long-time and multi-dimensional scanning and measurement. In 2011, Cuin et al. found in the study using non-invasive micro-test technique that Na” outflow capacity of a root system of a salt-tolerant wheat variety under salt stress is larger than that of a salt-sensitive wheat variety, but the K* outflow capacity is smaller than that of the salt-sensitive wheat variety.

[03] Plants absorb a large amount of Na’ under the salt stress to cause ion toxicity, meanwhile, absorption and accumulation of K* are inhibited to cause the reduction of the

K’/Na' ratio, and the content of Na” and K" in the plants under the salt stress can be used as an important index for evaluating the salt tolerance of the plants. At present, the identification of the salt tolerance of the cotton generally adopt salt pool, potted plant, seawater irrigation and other methods to measure physiological indexes such as cotton biomass and Na”, K content after salt treatment. The method not only occupies large area and is long in identification time, but also usually requires destructive sampling, and is sometimes unstable in effect. Therefore, it is necessary to establish a living, fast and accurate method for identifying the salt tolerance of cotton.

SUMMARY

[04] The object of the present disclosure is to solve the deficiencies of a conventional method for identifying salt tolerance of cotton, and provide a living, fast and accurate salt tolerance identification method suitable for plants including cotton.

[05] According to the technical scheme, a method for identifying salt tolerance of cotton, characterized in selecting robust cotton seedlings with 1 true leaf, treating with a nutrient solution containing 150 mM NaCl for 24 hours, selecting lateral roots with good growth state, shearing root tips, and pre-treating the root tips in a test buffer solution for 30 minutes; and then, fixing the root tips in a culture dish containing fresh test liquid, measuring by a using a non- invasive micro-test system, carrying out data analysis combined with Mageflux software to obtain the outflow velocity of Na” and K” of the root tips, and calculating Na*/K*. The larger the Na” outflow velocity, the smaller the K” outflow velocity, or the larger the Na*/K” flow velocity ratio, the stronger the salt tolerance; and on the contrary, the smaller the Na” outflow velocity, the larger the K* outflow velocity, or the smaller the Na’/K” velocity ratio, the worse 1

BL-5397 the salt tolerance.

LU501396

[06] The method specifically comprises the following steps:

[07] (1) Cotton seedling culture and salt treatment

[08] Selecting plump and full cotton seeds subjected to delinting, peeling off seed coats of the cotton seeds, disinfecting the cotton seeds for 15 min with a 0.1% mercuric chloride solution, washing for 6 times with sterilized double distilled water, planting on filter paper soaked with double distilled water (keeping moisture in the meantime), and germinating and emerging in a sunlight greenhouse (22-32 DEG C). After cotton seedling cotyledons are completely unfolded, selecting robust cotton seedlings with consistent size, putting in a seedling-raising pot of a

Hoagland nutrient solution for culture (22-32 DEG C, humidity of 60%-75%), and changing the nutrient solution every two days. After 1 true leaf grows, selecting the cotton seedlings with a complete root system and consistent size, and treating with the Hoagland nutrient solution containing 150 mM NaCl for 24 hours.

[09] (2) Na’, K” flow velocity measurement

[10] Selecting lateral roots with the same position, good growth condition and the same length and thickness for measurement of Na” and K* flow velocity. Specifically, shearing root tips of the lateral roots with scissors for (2-3 cm), washing with double distilled water, removing inorganic ions attached to the surface of the root tips, placing in a test buffer solution for balancing for 30 min (changing the buffer solution once or twice in the meantime), fixing the root tips in a culture dish containing fresh test liquid, and measuring the site of 0.5 distance from the root tips with a non-invasive micro-test system(NMT system BIO-IM); and three seedlings are measured for each cotton variety, two lateral roots are measured for each seedling, and each root is measured for 5-10 min;

[11] (3) Na’, K” flow velocity calculation and salt tolerance analysis

[12] Inputting original data obtained in step (2) into a Mageflux software for conversion to obtain Na’ and K* outflow velocity of the site of 0.5 cm distance from the root tips, and calculating Na™/K™. The larger the Na” outflow velocity, the smaller the K™ outflow velocity, or the larger the Na*/K* flow velocity ratio, the stronger the salt tolerance; and on the contrary, the smaller the Na” outflow velocity, the larger the K” outflow velocity, or the smaller the Na*/K* velocity ratio, the worse the salt tolerance.

[13] Compared with other methods, the method for identifying salt tolerance has the following advantages:

[14] (1) Data is accurate, and repeatability is good. Cotton seedling treatment is carried out under hydroponic conditions, the growth condition of the cotton root system before treatment is clear, the cotton seedlings with consistent growth can be selected, and the error of the test is reduced; the salt treatment time is short, and errors caused by too long salt treatment time are avoided; and advanced non-invasive micro-test technique can ensure accuracy and repeatability of the test.

[15] (2) Identification is fast. According to the method, the cotton seedlings cam be treated and measured just before and after one true leaf grows, so that the pre-culture time of the cotton seedlings is reduced; 24 hours after salt treatment, the ion flow velocity can be measured, the 2

BL-5397 salt treatment time is significantly shortened compared with other identification methods, and the Na”, K* flow velocity measurement of each line can be completed within a short time. LU501396

[16] (3) the non-invasive test is adopted. According to the method, no destructive sampling is needed, only two lateral roots are taken, normal growth of the tested cotton seedlings is not affected, and after test is completed, the cotton seedlings can continue to grow and develop or are used for other research; under a certain time, the vitality of the sheared lateral roots is the same as that of a living body root system, the Na”, K* flow velocity of the cotton root system in the living body state can be completely represented, and reaction and salt tolerance of the cotton plant under the growth state to the salt stress can be accurately displayed.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[17] The non-invasive micro-test system, the Mageflux software, the test buffer solution and the fresh test liquid for measuring Na”, K* flow velocity are provided by Xuyue (Beijing) Sci.

Tech. Co., Ltd.

Embodiment 1:

[18] Taking three sterilized culture dishes with a radius of 10 cm, placing two pieces of filter paper of the same size in the culture dishes, and adding 10 mL of sterilized double distilled water; taking 20 seeds each for declinated seeds of Simian 3 (SM3) and two salt-tolerant cotton lines (CMO2 and CMO4) that overexpress CMO gene of garden orache, soaking and peeling off seed coats, disinfecting the seeds with a 0.1% mercuric chloride solution for 15 min, then washing with sterilized double distilled water for 6 times, planting in the culture dishes correspondingly, and germinating and emerging in a greenhouse (22-32 DEG C). After cotton seedling cotyledons are completely unfolded, selecting ten robust cotton seedlings with consistent size for each variety, putting in a seedling-raising pot of a Hoagland nutrient solution for culture (22-32 DEG C, humidity of 60%-75%), and changing the nutrient solution every two days. After 1 true leaf grows, selecting three cotton seedlings with a complete root system and consistent size for each variety, and treating with the Hoagland nutrient solution containing 150 mM NaCl for 24 hours.

Table 1 Na” and K” flow velocity of SM3, CMO2, and CMO4 root systems after salt stress and ratio of Na+ and K+ flow velocity

Na" outflow (pmol/cm2s™) K* outflow (pmol/em”s"!)

[19] Selecting lateral roots with the same position, good growth condition and the same length and thickness, shearing root tips of the lateral roots with scissors for (2-3 cm), washing with double distilled water, placing in a test buffer solution for balancing for 30 min (changing the buffer solution once or twice in the meantime), fixing the root tips in a culture dish containing fresh test liquid by small stones and filter paper strips, and measuring Na” and K* flow velocity (original data is current value) by using a non-invasive micro-test system (NMT system BIO-IM). Measuring three seedlings for each cotton variety, and measuring two lateral roots for each seedling. Inputting original data obtained in step (2) into a Mageflux software, and calculating Na’ and K” outflow velocity (table 1) of SM3, CMO2 and CMO4 3

BL-5397 after salt stress. The Na” outflow of the transgenic salt tolerant cotton lines CMO2 and CMO4 under the salt stress is found to be increased by 27.5% and 25.9% respectively compared with LU501396 that of SM3, Na”/K" is increased by 44.8% and 39.9% respectively compared with that of SM3, but the K* outflow of CMO2 and CMO4 is decreased by 11.5% and 9.7% respectively compared with that of SM3. This is exactly consistent with the results that salt resistance of

CMO2 and CMO4 is significantly higher than that of SM3, illustrating that this technique can be used to identify salt tolerance of different cotton varieties.

Embodiment 2

[20] Taking two sterilized culture dishes with a radius of 10 cm, placing two pieces of filter paper of the same size in the culture dishes, and adding 10 mL of sterilized double distilled water; taking 20 full and plump seeds each for cotton (Lumianyan No.28)and Kosteletzkya virginica, peeling off seed coats, soaking with a 0.1% mercuric chloride solution for 15 min, then washing with sterilized double distilled water for 6 times, planting in the culture dishes correspondingly, and germinating and emerging in a greenhouse (22-32 DEG C). After cotton (Lumianyan No.28) and seedling cotyledons of Kosteletzkya virginica are completely unfolded, selecting 10 robust cotton seedlings with consistent size for each variety, putting in a seedling- raising pot of a Hoagland nutrient solution for culture (22-32 DEG C, humidity of 60%-75%), and changing the nutrient solution every two days. After the seedlings grow out of 1 true leaf, selecting three cotton seedlings with a complete root system and consistent size, and treating with the Hoagland nutrient solution containing 150 mM NaCl for 24 hours.

Table 2 Na”, K* flow velocity of Kosteletzkya virginica and Lumianyan No.28 root systems after salt stress and ratio of Na’ and K flow velocity. ; Na” outflow K" outflow (pmol/em<- Lt

[21] Selecting lateral roots with the same position, good growth condition and the same length and thickness, shearing root tips of the lateral roots with scissors (2-3 cm), washing with double distilled water, placing in a test buffer solution for balancing for 30 min (changing the buffer solution once or twice in the meantime), fixing the root tips in a culture dish containing fresh test liquid by small stones and filter paper strips, and measuring Na” and K” flow velocity (original data is current value) by using a non-invasive micro-test system (NMT system BIO-IM). Measuring three seedlings each for Lumianyan No.28 and

Kosteletzkya virginica, and measuring two lateral roots for each seedling. Inputting original data measured into a Mageflux software, and calculating Na” and K* outflow velocity and

Na*/K” (table 1) of Lumianyan No.28 and Kosteletzkya virginica root systems. The Na’ outflow of Kosteletzkya virginica under the salt stress is found to be 44.3% higher than with that of

Lumianyan No.28, Na’/K" is 88.9% higher than that of Lumianyan No.28, and K* outflow is 23.5% lower than that of Lumianyan No.28 (Table 2). This is exactly consistent with the results that salt resistance of Kosteletzkya virginica is significantly higher than that of Lumianyan

No.28, illustrating that this technique can be used to identify salt tolerance of plants. 4

Claims

BL-5397 WHAT IS CLAIMED IS: LU501396

1. A method for identifying salt tolerance of cotton, characterized in comprising the following steps of, firstly, selecting robust cotton seedlings with 1 true leaf, treating with a nutrient solution containing 150 mM NaCl for 24 hours, selecting lateral roots with good growth state, shearing root tips, and pre-treating the root tips in a test buffer solution for 30 minutes; and then, fixing the root tips in a culture dish containing fresh test liquid, measuring by a using a non-invasive micro-test system, carrying out data analysis combined with Mageflux software to obtain the outflow velocity of Na’ and K” of the root tips, and calculating Na/K*. The larger the Na © outflow velocity, the smaller the K © outflow velocity, or the larger the Na ‘/K * flow velocity ratio, the stronger the salt tolerance; and on the contrary, the smaller the Na” outflow velocity, the larger the K* outflow velocity, or the smaller the Na’/K” velocity ratio, the worse the salt tolerance.

2. The method for identifying salt tolerance of cotton according to claim 1, wherein (1) Cotton seedling culture and salt treatment Peeling off seed coats of cotton seeds, disinfecting the cotton seeds for 15 min with a 0.1% mercuric chloride solution, washing with sterilized double distilled water, planting on filter paper soaked with double distilled water, and germinating and emerging in a sunlight greenhouse; after cotton seedling cotyledons are completely unfolded, putting in a seedling- raising pot of a Hoagland nutrient solution for culture; after the seedlings grow out of 1 true leaf, selecting the cotton seedlings with a complete root system and consistent size, and treating with the Hoagland nutrient solution containing 150 mM NaCl for 24 hours; (2) Na”, K” flow velocity measurement Selecting lateral roots with good growth state, shearing root tips, washing with double distilled water, placing in a test buffer solution for 30 min, fixing the root tips in a culture dish containing fresh test liquid, and measuring the root tips with a non-invasive micro-test system; (3) Na”, K” flow velocity calculation and salt tolerance analysis Inputting original data obtained in step (2) into a Mageflux software for conversion to obtain Na ” and K * outflow velocity of the root tips, and calculating Na™/K".

3. The method for identifying salt tolerance of cotton according to any one of claims 1 to 2, wherein three seedlings are measured for each cotton variety, two lateral roots are measured for each seedling, and each root is measured for 5-10 min; The lateral roots for measurement are lateral roots with the same position, good growth condition and the same length and thickness, and the length of the root tips is 2-33 cm; The measurement site is at a distance of 0.5 cm from the root tips.