CN109618922B

CN109618922B - Screening method of peanut salt-tolerant ethylene insensitive mutant

Info

Publication number: CN109618922B
Application number: CN201910017036.3A
Authority: CN
Inventors: 乔利仙; 周文杰; 孙明明; 刘文平; 赵方贵; 朱虹; 隋炯明; 王晶珊
Original assignee: Qingdao Agricultural University
Current assignee: Qingdao Agricultural University
Priority date: 2019-01-08
Filing date: 2019-01-08
Publication date: 2022-04-08
Anticipated expiration: 2039-01-08
Also published as: CN109618922A

Abstract

The invention discloses a screening method of peanut salt-tolerant ethylene insensitive mutants, which utilizes a sand culture method; primarily screening ethephon; re-screening aminocyclopropane carboxylic Acid (ACC) which is an ethylene synthesis precursor; re-screening NaCl; culturing in dark, measuring the length of hypocotyl, and finally screening out the peanut salt-tolerant ethylene-insensitive mutant. The method has the advantages of being simple and capable of effectively screening the peanut salt-tolerant ethylene mutant.

Description

Screening method of peanut salt-tolerant ethylene insensitive mutant

Technical Field

The invention belongs to the technical field of biology, and relates to a screening method of peanut salt-tolerant ethylene insensitive mutants.

Background

Peanuts are important oil crops and economic crops in China and have an important position in agriculture and even the whole national economy. The salinization situation of the land in China is very severe, and the cultivation of new species of salt-tolerant crops is the most economic and effective way for utilizing the salinized soil. The method has the advantages that the genetic basis of the cultivated peanuts is narrow, high-salt-resistant germplasm is lacked in the existing resources, the salt-resistant mutants and the salt-resistant new germplasm are created and screened by a mutagenesis technology, the method has great significance for breeding the salt-resistant peanuts, and the method is an effective strategy for improving the salt resistance of the peanuts and improving the utilization efficiency of saline-alkali soil at present. 165 peanut salt-tolerant candidate germplasm is obtained by in vitro mutagenesis and salt-tolerant screening in earlier stage research, and the salt-tolerant mechanism of the germplasm is not researched. The salt-tolerant genes are discovered from the salt-tolerant germplasm and the salt-tolerant mechanism of the salt-tolerant germplasm is analyzed, so that the method has great significance for analyzing the salt-tolerant mechanism of the peanut and effectively utilizing the salt-tolerant germplasm. Ethylene is one of important plant hormones, not only regulates plant growth and development, but also plays an important role in plant adversity stress response. Ethylene participates in salt stress reaction at different levels and levels, the salt stress can induce the synthesis of ethylene in plants, and the ACC treatment of ethylene and ethylene precursors can also obviously increase the salt resistance and survival rate of the plants in a high-salt environment. Important components of the ethylene signaling pathway are also involved in salt stress responses in plants.

Disclosure of Invention

The invention aims to provide a screening method of peanut salt-tolerant ethylene insensitive mutants, which has the advantages of simple method and capability of effectively screening the peanut salt-tolerant ethylene mutants.

The technical scheme adopted by the invention is carried out according to the following steps:

step 1: a sand culture method is utilized;

step 2: primarily screening ethephon;

and step 3: 1-aminocyclopropane-1-carboxylic Acid (ACC), a precursor of ethylene synthesis, was rescreened;

and 4, step 4: re-screening NaCl;

and 5: culturing in dark, measuring the length of hypocotyl, and finally screening out the peanut salt-tolerant ethylene-insensitive mutant.

Further, 3 mg. L was used in step 2^-1And (3) carrying out seed germination treatment on the peanut germplasm obtained after mutagenesis by using ethephon solution.

Further, 50. mu. mol. L was used in step 3^-1And carrying out seed germination treatment on the selfed progeny of the peanut germplasm obtained by ethephon screening by using the ethylene synthesis precursor ACC.

Further, in the step 4, salt tolerance screening is carried out on the ethylene-insensitive germplasm obtained by screening the ethylene synthesis precursor ACC by adding 0.7% NaCl solution into sandy soil.

Drawings

FIG. 1 shows germinated peanut seeds after sand culture treatment;

FIG. 2 is a plot of "flowers 22" germinated seeds after ethephon treatment at various concentrations;

FIG. 3 is a comparison of hypocotyl length of "floral 22" treated with ethephon of different concentrations;

FIG. 4 shows a band width of 3 mg.L^-1Germinated peanut seeds after ethephon treatment;

FIG. 5 shows that the germplasm of different peanuts is 3 mg.L^-1Comparing the length of hypocotyls after ethephon solution treatment;

FIG. 6 is a "floral 22" germinated seed after treatment with different concentrations of ACC;

FIG. 7 is a comparison of hypocotyl length of "flower-raised 22" seedlings after ACC treatment at different concentrations;

FIG. 8 is germination of different peanut germplasm after ACC treatment;

FIG. 9 is a comparison of hypocotyl length of different peanut germplasms after treatment with an ACC solution;

FIG. 10 is a plot of "flowers grown 22" germinated seeds after treatment with different concentrations of NaCl;

FIG. 11 is a comparison of hypocotyl length of peanut seedlings treated with NaCl at different concentrations;

FIG. 12 is peanut germplasm after treatment with 0.7% NaCl solution;

FIG. 13 is a comparison of hypocotyl length of different peanut germplasms after treatment with NaCl solution.

Detailed Description

The present invention will be described in detail with reference to the following embodiments.

First, experimental material

165 peanut salt-tolerant candidate germplasm obtained by in vitro mutagenesis and salt-tolerant directional screening at the early stage and a mutagenic parent control variety 'flower culture 22'.

Second, Experimental methods

The invention comprises the following specific experimental steps:

1. determination of ethephon screening concentration

Washing off the soil adhered to the surface of the sand, rinsing with clear water for 2 times, placing in a beaker, sterilizing at high temperature and high pressure (121 ℃, 15min), and air drying. Adding 250mL of ethephon solution into 1000mL of sand, and stirring uniformly, wherein ethephon concentration gradient is set to be 1,3,5,7,10 mg.L^-1. Adding sand to the 500mL scale in the beaker, placing 10 peanut seeds on the surface of the sand, adding the sand to the 1000mL scale, and sealing the opening by using a preservative film. After dark culture in a culture room at 25 ℃ for 5 days, the sand on the upper layer of the beaker is poured off, the germinated and grown peanuts are taken out, and the hypocotyl length of the germinated and grown peanuts is measured (in figure 1, A: before ethephon treatment B: after 5 days of ethephon treatment). The assay was performed in 3 replicates and the data tested were analyzed for multiple comparisons using SPSS-21.0.

The test results show that different concentrations of ethephon solution treatment can inhibit the elongation of the hypocotyl of the peanut, and the inhibition effect is enhanced along with the increase of ethephon concentration (figures 2-3). The hypocotyl length of the seed without being treated by the ethephon solution is 6.24cm on average and 1 mg.L^-1The hypocotyl length after ethephon treatment was 3.06cm, and the difference between the two reached a very significant level (. P)<0.01)。3、5、7、10mg·L^-1The average length of hypocotyls of the ethephon solution treatment is 1.26, 1.16, 1.12 and 0.99cm, and the 4 concentration treatments have no significant difference with each other, but have the average length of 1 mg.L^-1Compared with the length of an embryonic axis of 3.06cm under the treatment of the ethephon solution, the differences reach a very significant level (. about.P)<0.01)。

Fig. 2 is a "flower-grown 22" germinated seed after ethephon treatment at different concentrations, where a: HY22 water treatment; b to F are 1,3,5,7,10 mg.L^-1And (4) carrying out ethephon treatment. FIG. 3 is a comparison of hypocotyl length (. X.P.) of "floral 22" treated with different concentrations of ethephon<0.01)；

To minimize selection errors, 3mg · L was finally determined^-1Ethephon concentration was used as the screening concentration for mutants.

2. Ethephon treatment for screening ethylene mutants

Use 3 mg.L^-1Ethephon solutionAnd (3) carrying out germination treatment, treatment and data analysis on 165 peanut salt-tolerant candidate germplasm in the same ethephon concentration screening method in the 1. The result shows that 145 germplasm shows that the growth of hypocotyl is inhibited, the thickening is shortened, and the germplasm belongs to normal ethylene sensitive germplasm. There were 11 germplasms (M29, M5, M4, M9, etc.) with no significant inhibition of hypocotyl growth (3 mg. L. in FIG. 4)^-1Germinated peanut seeds after ethephon treatment, wherein a: flower cultivation 22 water treatment; b: carrying out ethephon treatment on the flowers 22; c to F: part of the ethephon-treated ethylene-insensitive germplasm; g to H: the ethephon-treated partial ethylene hypersensitive germplasm) has the length higher than that of the hypocotyl of a control 'flower culture 22' treated under the same condition, and the difference reaches a very significant level (. about.P.. P.)<0.01) (fig. 5), belonging to ethylene insensitive germplasm. There were 9 germplasms (M8, M10, etc.) with significantly inhibited hypocotyl growth (fig. 4), with hypocotyl lengths significantly lower than those of "floral 22" treated under the same conditions and differences reaching very significant levels (. P. P.)<0.01) (figure 5 different peanut germplasm 3 mg. L^-1Comparison of hypocotyl Length after treatment with ethephon solution (. P)<0.01)) belonging to ethylene hypersensitive germplasm.

3 determination of screening concentration of ACC

Using 1, 10, 50. mu. mol. L^-1The ACC solution with the concentration gradient is used for germinating seeds 'florescence 22', processing an ethylene synthetic precursor aminocyclopropane-1-carboxylic Acid (ACC) and analyzing data by the same method as the ethephon screening method 1. The results show that the ACC solution treatments with different concentrations can inhibit the elongation of the peanut hypocotyl and the inhibition effect is enhanced along with the increase of the ACC concentration (figure 6 shows that the seeds germinated in 'flower culture 22' treated by ACC with different concentrations are treated by HY22 water, B-D: 1, 10 and 50 mu mol. L^-1ACC processing). The hypocotyl length of the seed without ACC solution treatment was 6.2cm on average and 10. mu. mol. multidot.L^-1The length of hypocotyl is 4.1cm after ACC solution treatment, and the difference reaches a very significant level (. about.P)<0.01)。50μmol·L^-1The average length of hypocotyls treated with ACC solution was 2.3cm, and 10. mu. mol. multidot.L^-1The difference of 4.1cm of hypocotyl length under ACC solution treatment reaches a very significant level (. P)<0.01) (FIG. 7 ACC treatment at different concentrationsComparison of hypocotyl Length of later "floral 22" seedlings (. P)<0.05,**.P<0.01). To minimize selection errors, 50. mu. mol. L was finally determined^-1ACC concentration was used as the screening concentration for mutants.

4 ACC treatment screening of ethylene mutants

50. mu. mol. L was used^-1The ACC solution carries out germination treatment on 11 parts of inbred progeny seeds of the ethylene-insensitive germplasm obtained by screening the ethephon solution, water treatment and ACC treatment are simultaneously set in contrast to 'flower cultivation 22', and the treatment and data analysis method is the same as that of the ethephon screening method 1.

The results showed that the hypocotyl growth of 9 germplasm was not significantly inhibited (FIG. 8 germination of different peanut germplasm treated with ACC, wherein A: HY22 water treatment; B-E: 50. mu. mol. L)^-1ACC treatment) with length higher than hypocotyl length of "floral 22" (wild type) under the same conditions and differences reached very significant levels (. P)<0.01) (FIG. 9 comparison of hypocotyl length (. P.) for different peanut germplasms treated with ACC solution<0.01)), it was preliminarily confirmed that the 9 ethylene insensitive mutants could be stably inherited.

Determination of 5 NaCl screening concentration

The seeds of 'flower culture 22' were germinated using NaCl solutions with concentration gradients of 0.1%, 0.5% and 0.7%, using the same ethod of ethephon concentration screening method 1. The results show that NaCl solutions with different concentrations all inhibit the elongation of the hypocotyl of the peanut, and the inhibition effect is enhanced along with the increase of the NaCl concentration (FIG. 10 shows that the 'flower culture 22' germinated seeds treated by NaCl with different concentrations are treated by HY22 water and B-D are treated by 0.1%, 0.5% and 0.7% NaCl). The hypocotyl length of the seed without NaCl solution treatment is 6.2cm on average, the hypocotyl length after 0.5% NaCl solution treatment is 3.8cm, and the difference between the two reaches a very significant level (. about.P < 0.01). The average length of hypocotyls treated with 0.7% NaCl solution was 2.2cm, respectively, and the difference was very significant (. P <0.01) compared to 3.8cm for hypocotyls treated with 0.5% NaCl solution (FIG. 11 comparison of hypocotyl lengths (. P <0.05,. P <0.01)) for peanut seedlings after treatment with different concentrations of NaCl. In order to reduce the selection error as much as possible, the 0.7% NaCl concentration is finally determined as the salt tolerance screening concentration of the mutant.

Salt-tolerant screening of 6 peanut ethylene-insensitive mutants

And (3) carrying out salt tolerance screening on 9 parts of ethylene-insensitive germplasm obtained by screening ACC by using 0.7% NaCl solution, and carrying out the same treatment and data analysis method as the ethephon concentration screening method 1. The results show that the hypocotyl length of 2 germplasm M29 and M5 is significantly higher than that of the control group 'floral 22' (fig. 12 peanut germplasm treated by 0.7% NaCl solution, A: HY22 water treatment; B-D: HY22, M29 and M5 treated by 0.7% NaCl), and the difference reaches a significant level (. P <0.01) (fig. 13 comparison of hypocotyl length of different peanut germplasm treated by NaCl solution (. P <0.05,. P <0.01)), and M29 and M5 are preliminarily confirmed to be peanut salt-tolerant ethylene insensitive mutants.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not intended to limit the present invention in any way, and all simple modifications, equivalent variations and modifications made to the above embodiments according to the technical spirit of the present invention are within the scope of the present invention.

Claims

1. A screening method of peanut salt-tolerant ethylene insensitive mutants is characterized by comprising the following steps:

step 1: a sand culture method is utilized;

step 2: primarily screening ethephon;

and step 3: re-screening aminocyclopropane carboxylic acid ACC serving as an ethylene synthesis precursor;

and 4, step 4: re-screening NaCl;

and 5: culturing in the dark, measuring the length of the hypocotyl, and finally screening out a peanut salt-tolerant ethylene-insensitive mutant;

wherein 3 mg.L is used in the step 2^-1Carrying out seed germination treatment on peanut salt-tolerant candidate germplasm obtained by in-vitro mutagenesis and salt-tolerant directional screening in the early stage by using a ethephon solution;

the step 3 uses 50 mu mol. L^-1Performing seed germination treatment on peanut germplasm selfing progeny obtained by ethephon screening by using an ethylene synthesis precursor aminocyclopropane carboxylic acid;

and 4, adding 0.7 percent NaCl solution into sandy soil, and performing salt tolerance screening on the ethylene-insensitive germplasm obtained by screening the ethylene synthesis precursor aminocyclopropane carboxylic acid.