Method for carrying out chemical emasculation by utilizing glyphosate potassium salt based on sea island cotton

Abstract

The invention provides a method for carrying out chemical emasculation by utilizing glyphosate potassium salt based on sea island cotton, which comprises the following steps of: adopting random block design, taking a glyphosate-resistant sea island cotton strain as a test material, and carrying out stem and leaf spraying on different block cotton by utilizing glyphosate potassium salt solutions with different concentration gradients in the early stage of budding; the method comprises the following steps: observing the phenotype of abortion of a sea island cotton plant treated by glyphosate potassium salt, carrying out cytological observation, detecting the activity of pollen grains, and counting the boll forming number in a harvesting period through a hybridization test, so as to screen out the optimal glyphosate potassium salt concentration capable of enabling the sea island cotton to be aborted. Therefore, the feasibility of preparing hybrids through chemical emasculation of the gossypium barbadense by utilizing the glyphosate potassium salt is identified. According to the method, the key period of glyphosate-induced male abortion of the gossypium barbadense and the appropriate glyphosate emasculation concentration can be determined, and the boll setting rate is higher than 80%, so that it is feasible that the gossypium barbadense is subjected to chemical emasculation by utilizing glyphosate to prepare the hybrid.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical male sterility in cotton, and particularly relates to a method for chemical male sterility based on Gossypium barbadense using potassium glyphosate. Background Art

[0002] Cotton is one of the most important economic crops in China. China is not only an important cotton producer, but also the most important cotton consumer and exporter of textile and clothing. Gossypium barbadense L, known for its "long, fine and strong" fibers and "smooth and soft" fabrics, is commonly known as long-staple cotton and is the "ceiling" of natural fiber quality. Gossypium barbadense has a wide range of uses. In the past, it was mainly used for military and special industries, and now it is widely used in civilian fabrics, playing an important role in improving people's clothing quality. Gossypium barbadense has strict requirements for light and heat conditions. In order to reverse the long-term dependence on foreign raw materials, China began to introduce Gossypium barbadense from Central Asia and other places in the early 1950s. At present, the Tarim Basin in Xinjiang Uygur Autonomous Region is the only Gossypium barbadense production area in China, with a total output accounting for about 30% of the world.

[0003] The Gossypium barbadense in Xinjiang Uygur Autonomous Region is known for its early maturity and excellent quality. Its maturity is about 20 days earlier than that of Gossypium barbadense in the United States and Egypt. Its fiber length can reach 40mm, specific strength is above 50cN / tex, fineness and regularity are good, and the color is white, filling the gap that China cannot produce Gossypium barbadense. In recent years, high-yield, high-resistance and high-quality cotton varieties have been obtained through cross-breeding, improving cotton yield and economic benefits. However, artificial hybridization is time-consuming and laborious, and the boll setting rate cannot be fully guaranteed. This high-cost seed production technology restricts the development of cross-breeding. Therefore, improving the cross-breeding seed production technology is a potential factor for increasing cotton yield and effectively promoting the development of the cotton industry. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for chemical male sterility based on Gossypium barbadense using potassium glyphosate in view of the deficiencies of the above-mentioned prior art. This method can determine the critical period of male sterility induced by glyphosate and the appropriate concentration of glyphosate for emasculation in Gossypium barbadense. If the boll setting rate is higher than 80%, it indicates that it is feasible to use glyphosate for chemical male sterility in Gossypium barbadense to prepare hybrid seeds.

[0005] To solve the above technical problem, the technical solution adopted by the present invention is: a method for chemical male sterility based on Gossypium barbadense using potassium glyphosate, and the method is as follows:

[0006] S1. Adopt a randomized block design, use a glyphosate-resistant Gossypium barbadense line as the experimental material. After the cotton emerges, perform artificial thinning. Before the pre-budding stage, spray the stems and leaves of cotton in different blocks with potassium glyphosate solutions at different concentration gradients to obtain Gossypium barbadense plants treated with potassium glyphosate.

[0007] The glyphosate potassium salt solution is prepared by diluting with water the glyphosate potassium salt technical material with a total active ingredient content of 95% produced by Jiangxi Jinlong Chemical Industry Co., Ltd.;

[0008] S2. Observe the phenotypic, cytological observations of the abortion of the Gossypium barbadense plants treated with glyphosate potassium salt in S1 and detect the pollen grain activity, determine the critical period of the abortion of Gossypium barbadense caused by glyphosate potassium salt and whether Gossypium barbadense can use glyphosate for chemical male sterility to assist in the preparation of hybrid seeds;

[0009] S3. Screen the optimal concentration of glyphosate potassium salt through the number of bolls set during the hybrid test harvest period, and thus identify the feasibility of using glyphosate potassium salt for chemical male sterility to prepare hybrid seeds of Gossypium barbadense.

[0010] Preferably, the concentrations of the glyphosate potassium salt solutions with different concentration gradients in S1 are 3.75 g / L, 7.5 g / L, 15 g / L, and 30 g / L. At the same time, a water control is set, and each treatment is set with three replicates.

[0011] Preferably, the method for observing the phenotypic abortion of the Gossypium barbadense plants in S2 is as follows: After treating with the glyphosate potassium salt solutions with different concentration gradients for 10 days, observe the color change of the anthers in the young buds of Gossypium barbadense to judge the male abortion of Gossypium barbadense; after treating with the glyphosate potassium salt solutions with different concentration gradients for 10 days, observe the phenotype and female organs.

[0012] Preferably, the method for cytological observation of the abortion of the Gossypium barbadense plants in S2 is as follows: After sampling the anthers of Gossypium barbadense treated with the glyphosate potassium salt solutions with different concentration gradients at different times, conduct stereoscopic microscopy observations.

[0013] Preferably, the sampling time of the anthers is the 5th day, 10th day, 15th day, and 20th day after treatment with the glyphosate potassium salt solution; compare the anthers treated with the glyphosate salt solution at different times with the water control.

[0014] Preferably, the method for detecting the pollen grain activity in S2 adopts the TTC method of the pollen viability detection kit, and after sampling the pollen of Gossypium barbadense treated with the glyphosate potassium salt solutions with different concentration gradients at different times, conduct viability detection.

[0015] Preferably, the sampling time of the pollen is the 3rd day, 5th day, 6th day, 7th day, 8th day, and 9th day after treatment with the glyphosate potassium salt solution; compare the pollen treated with the glyphosate salt solution at different times with the water control.

[0016] Preferably, the specific method of the hybridization test in S3 is as follows: The hybridization experiment is carried out at the full-bloom stage 30 days after the treatment with the potassium glyphosate solution. Select the normal flowers in the control to pollinate the treatment groups treated with different concentrations of potassium glyphosate solution. Make 30 hybrid bolls for each treatment, and investigate the number of set bolls in each treatment group during the harvest period.

[0017] Preferably, through the number of set bolls in each treatment group during the harvest period, determine the critical period of male sterility induced by potassium glyphosate in sea island cotton and the suitable emasculation concentration of potassium glyphosate. When the boll setting rate > 80%, it indicates that it is feasible to use glyphosate for chemical emasculation of sea island cotton to prepare hybrid seeds.

[0018] The present invention has the following advantages compared with the prior art:

[0019] The present invention can determine the critical period of male sterility induced by glyphosate in sea island cotton and the suitable emasculation concentration of glyphosate. When the boll setting rate is higher than 80%, it indicates that it is feasible to use glyphosate for chemical emasculation of sea island cotton to prepare hybrid seeds.

[0020] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Detailed implementation manners

[0021] Example 1

[0022] The method for chemical emasculation of sea island cotton using potassium glyphosate according to the present invention is as follows:

[0023] S1. Adopt a randomized block design. Use the glyphosate-resistant sea island cotton line as the test material. After the cotton seedlings emerge, conduct artificial thinning. Before the pre-bud stage, spray the cotton in different blocks with potassium glyphosate solutions at different concentration gradients on the stems and leaves to obtain the sea island cotton plants after potassium glyphosate treatment;

[0024] The glyphosate-resistant sea island cotton line is the long-staple cotton variety K426 line;

[0025] The potassium glyphosate solution is prepared by diluting the potassium glyphosate technical with a total active ingredient content of 95% produced by Jiangxi Jinlong Chemical Industry Co., Ltd. with water;

[0026] S2. Observe the phenotypic, cytological observations of the sterility of the sea island cotton plants after potassium glyphosate treatment obtained in S1 and detect the pollen grain activity to determine the critical period of potassium glyphosate causing sterility in sea island cotton and whether sea island cotton can use glyphosate for chemical emasculation to assist in preparing hybrid seeds;

[0027] S3. Screen the optimal potassium glyphosate concentration through the number of set bolls during the harvest period of the hybridization test to identify the feasibility of using potassium glyphosate for chemical emasculation of sea island cotton to prepare hybrid seeds.

[0028] In this example, the concentrations of the glyphosate potassium salt solutions with different concentration gradients in S1 are 3.75 g / L, 7.5 g / L, 15 g / L, and 30 g / L. Meanwhile, a water control is set, and each treatment is set with three replicates.

[0029] In this example, the method for observing the phenotypes of the abortive Gossypium barbadense plants in S2 is as follows: After treating with glyphosate potassium salt solutions with different concentration gradients for 10 days, observe the color change of anthers in the young buds of Gossypium barbadense to judge male abortion; after treating with glyphosate potassium salt solutions with different concentration gradients for 10 days, observe the phenotypes and female organs.

[0030] In this example, the method for cytological observation of the abortive Gossypium barbadense plants in S2 is as follows: After sampling the anthers of Gossypium barbadense at different stages treated with glyphosate potassium salt solutions with different concentration gradients, conduct stereoscopic microscopy observation; the sampling time of the anthers is the 5th, 10th, 15th, and 20th days after treatment with glyphosate potassium salt solutions; compare the anthers treated with glyphosate salt solutions at different stages with the water control.

[0031] In this example, the TTC method of the pollen viability detection kit is used to detect the pollen grain viability. After sampling the pollen of Gossypium barbadense at different stages treated with glyphosate potassium salt solutions with different concentration gradients, conduct viability detection; the sampling time of the pollen is the 3rd, 5th, 6th, 7th, 8th, and 9th days after treatment with glyphosate potassium salt solutions; compare the pollen treated with glyphosate salt solutions at different stages with the water control.

[0032] In this example, after treating with the glyphosate potassium salt solution for 15 days, sample according to the bud length. Fix the anthers taken from the range of 2 - 3 mm to 23 - 24 mm, and then conduct paraffin sectioning later. The specific method is as follows:

[0033] (1) Fix the buds in pre-cooled FAA. The volume of the fixing solution is more than 10 times that of the tissue. Vacuumize on an ice pack for 15 min, then slowly release the air, repeat the vacuumization twice until the buds sink to the bottom, and then replace the fixing solution and place it in a 4℃ refrigerator overnight.

[0034] (2) Then dehydrate using graded ethanol (50 - 95%), then dehydrate twice with absolute ethanol at an interval of 2 hours, and then transfer to gradient absolute ethanol and xylene (in three times, one hour each time). Repeat twice in xylene, one hour each time. Then transfer to gradient xylene and paraffin (in three times, one hour each time). Finally, repeat twice in paraffin, one day each time.

[0035] (3) Embed at room temperature, obtain 12 - μm - thick sections with a rotary microtome. Subsequently, place the sections on a glass slide coated with polylysine after drying and dry them on a spreading machine. Then, dewax, rehydrate, and stain, and then dehydrate, clear, and mount the slides.

[0036] Samples were taken from different fresh parts (fresh young buds, ovaries, leaves, anthers, petals, bracts, and shoot tips) of Gossypium barbadense plants treated with different concentrations, and after thorough grinding, gene expression levels, shikimic acid, and EPSPS synthase were detected. For the detection of gene expression levels, RNA was extracted using a kit, followed by reverse transcription, and finally qRT-PCR was performed. Spectrophotometry was used for the detection of shikimic acid and EPSPS synthase.

[0037] In this example, the specific method of the hybridization experiment described in S3 is as follows: The hybridization experiment was carried out at the full flowering stage 30 days after treatment with the potassium glyphosate solution. Normal flowers in the control were selected to pollinate the treatment groups treated with different concentrations of potassium glyphosate solution. 30 hybrid bolls were made for each treatment, and the number of set bolls in each treatment group was investigated at the harvest stage.

[0038] In this example, the critical period of male sterility induced by potassium glyphosate in Gossypium barbadense and the suitable emasculation concentration of potassium glyphosate were determined based on the number of set bolls in each treatment group at the harvest stage. When the boll setting rate > 80%, it indicates that it is feasible to use glyphosate for chemical emasculation of Gossypium barbadense to prepare hybrid seeds.

[0039] The above is only a preferred embodiment of the present invention and does not impose any limitation on the present invention. Any simple modification, change, and equivalent change made to the above embodiments based on the technical essence of the invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A method for chemical male sterilization of sea island cotton using potassium glyphosate, characterized in that: The method is: S1. A randomized block design was adopted, and glyphosate-resistant sea island cotton strains were used as test materials. After the cotton seedlings emerged, the seedlings were manually determined. In the early stage of bud formation, different concentrations of glyphosate potassium salt solutions were used to spray the stems and leaves of cotton in different blocks to obtain sea island cotton plants treated with glyphosate potassium salt. The glyphosate potassium salt solution is prepared by diluting the glyphosate potassium salt original medicine with a total active ingredient content of 95% produced by Jiangxi Jinlong Chemical Co., Ltd. with water; S2, observing the phenotype of the aborted sea island cotton plants after the treatment with potassium glyphosate obtained in S1, conducting cytological observation and detecting the activity of pollen grains, determining the critical period when potassium glyphosate causes the sea island cotton to abort and whether glyphosate can be used to chemically sterilize the sea island cotton to assist in the preparation of hybrids; S3. The optimal glyphosate potassium concentration was screened by the number of bolls at the harvest stage of the hybridization experiment to identify the feasibility of using glyphosate potassium to chemically sterilize male seeds in sea island cotton to prepare hybrids.

2. The method for chemical male sterilization based on sea island cotton using glyphosate according to claim 1, characterized in that: The concentrations of the glyphosate potassium salt solutions with different concentration gradients described in S1 are 3.75 g / L, 7.5 g / L, 15 g / L, and 30 g / L. A clear water control is also set, and each treatment is repeated three times.

3. The method for chemical male sterilization based on sea island cotton using glyphosate according to claim 1, characterized in that: The method for observing the phenotype of sterility of the Sea Island cotton plants described in S2 is: after being treated with glyphosate potassium salt solutions of different concentration gradients for 10 days, the color changes of anthers in the young buds of the Sea Island cotton are observed to determine the male sterility of the Sea Island cotton; after being treated with glyphosate potassium salt solutions of different concentration gradients for 10 days, the phenotype and female organs are observed.

4. The method for chemical male sterilization based on sea island cotton using glyphosate according to claim 1, characterized in that: The method for cytological observation of plant abortion in Sea Island Cotton described in S2 is: sampling anthers of Sea Island Cotton at different stages after being treated with glyphosate potassium salt solutions of different concentration gradients and then observing under a stereomicroscope.

5. The method for chemical male sterilization based on sea island cotton using glyphosate according to claim 4, characterized in that: The sampling time of the anthers is 5 days, 10 days, 15 days and 20 days after the treatment with the glyphosate potassium salt solution; the anthers treated with the glyphosate salt solution at different periods are compared with the clear water control.

6. The method for chemical male sterilization based on sea island cotton using glyphosate according to claim 1, characterized in that: The pollen activity detection described in S2 adopts the pollen viability detection kit TTC method, and pollen samples of sea island cotton at different stages treated with glyphosate potassium salt solutions of different concentration gradients are sampled and then viability detection is performed.

7. The method for chemical male sterilization using glyphosate based on sea island cotton according to claim 6, characterized in that: The pollen was sampled on the 3rd, 5th, 6th, 7th, 8th and 9th day after being treated with the glyphosate potassium salt solution; the pollen treated with the glyphosate salt solution at different times was compared with the clear water control.

8. The method for chemical male sterilization using glyphosate based on sea island cotton according to claim 1, characterized in that: The specific method of the hybridization test described in S3 is: a hybridization experiment is carried out at the peak flowering period 30 days after treatment with glyphosate potassium salt solution, normal flowers in the control group are selected to pollinate the treatment groups treated with glyphosate potassium salt solution of different concentrations, 30 hybrid bolls are made for each treatment, and the number of bolls in each treatment group is investigated at the harvest period.

9. The method for chemical male sterilization using glyphosate based on sea island cotton according to claim 8, characterized in that: The critical period of male sterility induced by glyphosate potassium and the appropriate emasculation concentration of glyphosate potassium were determined by the number of bolls in each treatment group at the harvest time. When the boll setting rate was greater than 80%, it indicated that it was feasible to use glyphosate to chemically sterilize males in sea island cotton to prepare hybrids.