CN113498734A - Non-transgenic cotton breeding method - Google Patents
Non-transgenic cotton breeding method Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
- A01H1/021—Methods of breeding using interspecific crosses, i.e. interspecies crosses
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
Abstract
The invention belongs to the technical field of cotton breeding, and particularly relates to a non-transgenic cotton breeding method. Crossing the local non-transgenic cotton variety obtained by screening as female parent and the externally introduced non-transgenic cotton variety as male parent in a non-transgenic cotton breeding test field to obtain F0Generation hybrid bells; f is to be0The generation hybrid bolls are continuously selfed for 2 times in a non-transgenic cotton breeding test field, and then F is selected2Inbreeding generation individual plant in non-transgenic cotton breeding test field to obtain F3Planting and transplanting F3The generation plants are planted in a non-transgenic cotton breeding test field for selfing to obtain F4And (4) generating strains, and obtaining the strains of the non-transgenic cotton after identification and screening by using the generation strains. The breeding method of the invention separately establishes the non-transgenic cotton breeding test field, and has a 100-200m isolation zone with the transgenic cotton test field, thereby ensuring the purity of the non-transgenic cotton in space.
Description
Technical Field
The invention belongs to the technical field of cotton breeding, and particularly relates to a non-transgenic cotton breeding method.
Background
Cotton is the main economic crop in China. The single yield and the total yield of cotton in China are the first in the world. In recent years, the area of cotton in China is more than 5000 ten thousand mu. The Xinjiang light, heat, water and soil resources are rich, the cotton-suitable area is wide, the planting area of Xinjiang cotton is more than 3000 ten thousand mu in recent years, and the single yield and the total yield of the Xinjiang cotton are the first of the cotton planting provinces (autonomous regions) in China. Xinjiang is also the main production area of high-quality cotton with the most development potential in China, wherein the early-maturing cotton planting area in northern Xinjiang has about 800 ten thousand mu of cotton field, is one of the important cotton planting areas in Xinjiang, is also an ecological area with unique and thick planted high-quality upland cotton, and the high-quality raw cotton produced in the ecological area accounts for more than 80 percent of the export cotton in China and is the most main export cotton base in China and Xinjiang. As the continuous cropping age of cotton fields is prolonged, the blight and verticillium wilt of cotton also rapidly spread, and the blight and verticillium wilt become a main obstacle for continuous high and stable yield of cotton. At present, more than 80% of cotton fields in Xinjiang are damaged by blight and verticillium wilt. Blight and verticillium wilt are one of the main factors restricting the production and development of Xinjiang cotton. Xinjiang requires a new variety of cotton with precocity, disease resistance, high yield (more than 40% of coat, medium leaf size and moderate plant type) and high quality. According to the characteristics of good prematurity and outstanding disease resistance of the Liaoling cotton variety, the test demonstration and popularization of the prematurity disease-resistant high-yield high-quality Liaoling cotton variety have wide prospects in areas with the growth period of about 125 days, such as Usu in northern Xinjiang, Kutun, Shawan, Shihe, Manasi and the like.
At present, Xinjiang is a nationally limited non-transgenic cotton planting area, and the varieties participating in regional tests of Xinjiang autonomous cotton varieties are mainly non-transgenic cotton varieties. However, the non-transgenic cotton variety planted at the present stage has the defects of poor disease resistance and late maturity, so the method for cultivating the non-transgenic cotton variety with good disease resistance, early maturity, high yield and high quality has important significance.
Disclosure of Invention
The invention provides a non-transgenic cotton breeding method, which aims to solve the problem that most of the cotton varieties are transgenic insect-resistant cotton varieties at present and are difficult to obtain new non-transgenic cotton varieties, and cultivate non-transgenic cotton varieties suitable for being planted in northern Xinjiang.
In order to achieve the above object, the method provided by the present invention specifically comprises the following steps:
a non-transgenic cotton breeding method includes such steps as screening to obtain local non-transgenic cotton variety as female parent, introducing the non-transgenic cotton variety as male parent, and hybridizing in the test field to obtain F0Generation hybrid bells; f is to be0The generation hybrid bolls are continuously selfed for 2 times in a non-transgenic cotton breeding test field, and then F is selected2Inbreeding generation individual plant in non-transgenic cotton breeding test field to obtain F3Planting and transplanting F3The generation plants are planted in a non-transgenic cotton breeding test field for selfing to obtain F4And (4) generating strains, and obtaining the strains of the non-transgenic cotton after identification and screening by using the generation strains.
Further, F0Continuously identifying and screening the generation hybrid bolls in a non-transgenic cotton breeding test field for 4 years to obtain stable F meeting the breeding target (the growth period is less than or equal to 125 days, the yield of 5 m-row long-seed cotton is more than or equal to 0.9kg, the average length of the upper half part of fiber is more than or equal to 29.0mm, the breaking specific strength is more than or equal to 30.0cN/tex, and the micronaire value is 3.5-5.0)4Generation line, F obtained4And (4) entrusting the seeds of the generation line to the ' plant ecological environment safety supervision and inspection test center ' of the department of agricultural rural areas ' to detect, and obtaining the line of the non-transgenic cotton when the seeds are qualified.
F eligible for detection (i.e., non-transgenic)4The next year of the strain (F)5Generations) to carry out strain comparison test and survey and identify fusarium wilt and verticillium wilt. Selecting good F5The generation strain (growth period is less than or equal to 125 days, the yield of the ginned cotton is more than or equal to 7 percent compared with the area test control variety of Xinjiang (Liaoning), the wilt disease is less than or equal to 10 percent, the greensickness disease is less than or equal to 20 percent, the average length of the upper half part of the fiber is more than or equal to 29.0mm, the breaking ratio strength is more than or equal to 30.0cN/tex, and the micronaire value is 3.5-5.0) for the next year (F6Generation strain) is subjected to different-place multipoint sample ratio test, then enters into legal test (namely, Liaoning province cotton variety regional test or Xinjiang autonomous region cotton variety regional test), passes through the legal test (Liaoning province or Xinjiang autonomous region cotton variety regional test and production testTest), and obtaining (the crop variety approval committee in Liaoning province or Xinjiang autonomous region) to approve to obtain a new non-transgenic cotton variety.
The technical indexes of the variety are as follows:
(1) the growth period indexes are as follows: the super-early-maturing cotton area of Liaoning, etc. is less than or equal to 125 days;
(2) the fiber quality index is as follows: type I: the average length of the upper half part of the fiber is more than or equal to 31mm, the breaking specific strength is more than or equal to 32cN/tex, and the micronaire value is 3.7-4.2; type II: the average length of the upper half part of the fiber is more than or equal to 29mm, the breaking specific strength is more than or equal to 30cN/tex, and the micronaire value is 3.5-5.0; type III: the average length of the upper half part of the fiber is more than or equal to 27mm, the breaking specific strength is more than or equal to 28cN/tex, and the micronaire value is 3.5-5.5.
(3) The yield index is as follows: type I: the yield of the ginned cotton is increased by more than or equal to 0.0 percent compared with the control variety of the test in the Xinjiang (Liaoning) area; type II: the yield of the ginned cotton is increased by more than or equal to 7.0 percent compared with the control variety of the test in the Xinjiang (Liaoning) area; type III: the yield of the ginned cotton is increased by more than or equal to 10.0 percent compared with the control variety of the test in the Xinjiang (Liaoning) area. The clothes score reaches more than 40 percent; the blooming rate is more than or equal to 85 percent.
(4) Disease resistance index: the wilt is less than or equal to 15, and the verticillium wilt is less than or equal to 35.
The female parent and the male parent are screened in such a way that non-transgenic local cotton varieties and externally introduced cotton resource seeds are subjected to non-transgenic detection by a Bt gene transfer test strip, the seeds of the test strip display detection line are cotton seeds containing the Bt gene transfer, and the seeds of the test strip which do not display the detection line are cotton seeds not containing the Bt gene transfer, namely non-transgenic cotton seeds, and are used as a hybrid female parent and a male parent.
Respectively and independently planting the screened female parent seeds and the screened male parent seeds in a non-transgenic cotton breeding test field, spraying kanamycin sulfate aqueous solution in a seedling stage (2-4 true leaves), and keeping cotton seedlings with yellow leaves (namely, non-transgenic cotton seedlings); pulling out the cotton seedlings which are not discolored (namely green). The remained non-transgenic cotton seedlings grow and develop, blossom and bear bolls, and seeds are harvested in autumn for later use.
The concentration of the kanamycin sulfate in the kanamycin sulfate aqueous solution is 3000 mg/kg.
Non-transgenic cotton seeds obtained after laboratory detection are planted in a non-transgenic cotton breeding test field and are repeatedly identified by an aqueous solution containing kanamycin sulfate in the seedling stage, then larger cotton seedlings with yellow cotyledons or true leaves are reserved, smaller cotton seedlings which are not yellow (namely green) and yellow are pulled out, the reserved larger yellow cotton seedlings grow and develop, blossom and bear bolls, and seeds harvested in autumn are used as breeding parents.
The female parent and the male parent are hybridized after screening, that is, in the full-bloom stage of the cotton (namely, 19-24 days in 7 months), sunny weather is selected, and at 2-4 pm on the day, the stamens of the female parent are stripped off, and the stigma (namely, pistil) is reserved; picking flower of male parent at 9-11 am on the next day, smearing pollen of stamen of flower of male parent on stigmas of flower of female parent, and pollinating cotton boll developed and grown successfully to obtain F0Generation hybrid bells.
The first part is0Planting the generation hybrid bolls in a non-transgenic cotton breeding test field to obtain F1Selecting excellent F with growth period not more than 125 days, 5 m seed cotton yield not less than 0.9kg, fiber upper half average length not less than 29.0mm, breaking specific strength not less than 30.0cN/tex and micronaire value 3.5-5.01Combining the generations; will be excellent F1Planting the generation combination in a non-transgenic cotton breeding test field in the next year to obtain F2Selfing combination, selecting excellent F in autumn2Generation individual plants (growth period is less than or equal to 125 days, average length of the upper half part of the fiber is more than or equal to 29.0mm, breaking ratio strength is more than or equal to 30.0cN/tex, and micronaire value is 3.5-5.0); in the third year, good F will be selected2Planting the single generation plant in a non-transgenic cotton breeding test field to obtain F3Plant generation and selection of excellent F in autumn3The plant generation row (the growth period is less than or equal to 125 days, the yield of the seed cotton growing in 5 m row is more than or equal to 0.9kg, the average length of the upper half part of the fiber is more than or equal to 29.0mm, the breaking specific strength is more than or equal to 30.0cN/tex, and the micronaire value is 3.5-5.0); in the fourth year, F is selected3Planting the plant generation row in a non-transgenic cotton breeding test field to obtain F4Generation line (i.e. multirow F)4Generation), selecting excellent F in autumn4The generation strain (the growth period is less than or equal to 125 days, the yield of the seed cotton growing in 5 m rows is more than or equal to 0.9kg, the average length of the upper half part of the fiber is more than or equal to 29.0mm, the breaking specific strength is more than or equal to 30.0cN/tex, and the micronaire value is 3.5-5.0).
The local cotton varieties are Liaoling cotton No. 18, Liaoling cotton No. 25 and Liaoling cotton No. 16; the cotton resource introduced from the outside includes New Luzao No. 10, New Luzao No. 17, New Luzao No. 61 and New Luzao No. 62.
Screening non-transgenic local cotton varieties and externally introduced resources in a laboratory: firstly, carrying out non-transgenic detection on non-transgenic local cotton varieties and externally introduced cotton resource seeds in a laboratory by using a Bt gene transfer test strip, wherein the seeds in a test strip display detection line are cotton seeds containing the Bt gene transfer, and the seeds in a test strip non-display detection line are cotton seeds not containing the Bt gene transfer, namely non-transgenic cotton seeds. Wherein the Bt gene transfer test strip is a gold-labeled Bt-CryIAb/Ac test strip (the detection precision is 0.125 percent), and the dilution ratio of the seeds is 1: 20.
The non-transgenic cotton breeding experimental field comprises: an independent non-transgenic cotton breeding test field and a transgenic cotton test field are set with a 100-200m isolation zone.
Compared with the prior art, the invention has the following advantages:
1. according to the breeding method, the non-transgenic cotton seeds are detected by using the Bt-transgenic test strip with the detection precision of 0.125%, and the purpose of strict screening is achieved in the first step. This step highlights the unexpected benefits of the present application.
2. The breeding method of the invention separately establishes the non-transgenic cotton breeding test field, and has a 100-200m isolation zone (the isolation zone of the unit also has trees and houses) with the transgenic cotton test field, thereby ensuring the purity of the non-transgenic cotton planting in space. The step also highlights the innovation point of the invention, because in cotton breeding, a transgenic cotton breeding test field is generally established independently.
3. The breeding method of the invention utilizes kanamycin sulfate to continuously identify the parents and 4 generations of filial generations in the seedling stage, thereby further ensuring the purity of non-transgenic cotton varieties. The step is also innovative, and the identification is mainly carried out by using kanamycin sulfate in the seedling stage, namely a method for identifying transgenic cotton.
4. According to the breeding method, the parent material and the filial generation seedling head line entrust the national authority to carry out non-transgenic detection, so that the purity of the selected non-transgenic cotton variety is comprehensively ensured. The method has unique innovation of entrusting national authorities to perform non-transgenic detection on the parent material in the step, and generally entrusts the national authorities to perform non-transgenic detection on the seedling head strain.
Drawings
FIG. 1 is a schematic diagram of a non-transgenic cotton breeding method according to an embodiment of the present invention.
FIG. 2 is a picture of a laboratory Bt transgenic test strip for detecting non-transgenic cotton seeds according to an embodiment of the present invention; wherein, the left detection cup is a Bt gene-transferred cotton seed solution, and a black detection line appears below the test strip; the right detection cup is non-transgenic Bt cotton seed liquid, and no black detection line appears below the test strip.
FIG. 3 is a yellow picture of leaves of a non-transgenic cotton seedling provided by an embodiment of the present invention after kanamycin sulfate is sprayed. Note: in the figure, cotton seedlings with yellow leaves are non-transgenic cotton seedlings.
Detailed Description
The present invention is described in detail below with reference to specific examples, but the present invention should not be construed as being limited thereto. The test methods in the following examples, which are not specified in specific conditions, are generally conducted under conventional conditions, and the steps thereof will not be described in detail since they do not relate to the invention.
Liaomian No. 18: the method is recorded in the Chinese cotton, 2003, (10): 33-34 ".
New luohao No. 17: the contents of.
Liaomian No. 16: it is reported in "New Cotton variety, Liaomian No. 16. Wangdsheng. Chinese agricultural technology popularization, year 2000, stage 05".
Liaomian No. 25: the method is recorded in new agriculture of New Aphanotis Kagaku No. 25, Wangzheng, Gaoying, Wangwei Wei and the like, and 004 stage of 2012 (000 rolls).
Xinluzao No. 10: is recorded in Xinjiang agricultural science and technology, No. 2003 (000 volumes) at 001 stage 14, of 'New Luzao Zao No. 19' Zhengzhilong, cheap Euro Yang and Yuanjiachang 'of disease-resistant super-early-maturing cotton variety'.
New luohao No. 61: the method is recorded in the research on the change of soil moisture and nutrients in different periods of a Xinluzao No. 61 drip irrigation cotton field, Zhao Hai, Zhang Yan, Wanggang and the like, cotton science, and the 003 period of 2017 (039 volume) which is 4 pages in total.
New Luzao No. 62: described in "breeding and cultivating technology of New variety of early-maturing upland cotton, New Luzao No. 62, Qinjing hong, Yangyangyong forest, Wanying, etc., crop J, 2014 1.
Liaomian 34: the description is as follows: "breeding and cultivating technology of Liaomian 34 cotton as new variety of super-early-maturing high-yield and high-quality" Liu Yan Zhen, Cao Yan, single crystal, etc. Chinese cotton, No. 44, No. 10 of 2017: 22-23 ".
The non-transgenic cotton breeding method (figure 1) of the invention comprises the following steps:
1. laboratory screening of non-transgenic local cotton varieties and external introduction of resources
And detecting the non-transgenic local cotton variety and the externally introduced cotton resource by using a gold-labeled Bt-CryIAb/Ac test strip (the detection precision is 0.125%). Pulverizing the seeds into powder by a pulverizer, diluting the seeds by distilled water at a ratio of 1:20(0.1ml/2ml), inserting the test strip into a liquid cup, and taking the cotton seeds without detection lines as non-transgenic seeds.
2. Single-construction non-transgenic cotton breeding experimental field
And a 100-200m isolation zone is arranged with the transgenic cotton test field, and a non-transgenic cotton breeding test field is independently established, so that the purity of non-transgenic cotton breeding is ensured in space.
3. Identification of kanamycin sulfate in seedling stage
Planting a non-transgenic local cotton variety and an externally introduced resource detected from a laboratory in a non-transgenic cotton breeding test field, selecting high-temperature sunny weather, spraying a kanamycin sulfate solution with the concentration of 3000mg/kg water in the stage that cotton seedlings grow 2-4 true leaves, and investigating after 7-10 days. The cotton seedling with the yellow color of the true leaf and the cotyledon is a non-transgenic cotton seedling; the cotton seedling which does not turn yellow (namely is still green) is the Bt gene transfer insect-resistant cotton, and the cotton seedling is required to be completely pulled out. Then, the rest yellow (non-transgenic) cotton seedlings are subjected to final singling (namely, 1 big seedling and strong seedling is reserved in each hole).
4. National center of qualification testing
In autumn, the harvested non-transgenic local cotton variety and the harvested externally introduced resource seeds are entrusted to the center for plant ecological environment safety supervision and inspection testing in rural areas for detection, and the reliability of the harvested non-transgenic seeds is further identified.
5. Crossbreeding
And (3) continuously planting the non-transgenic local cotton variety and the externally introduced resource which are qualified by the detection of the plant ecological environment safety supervision inspection test center of the department of agricultural rural areas in a non-transgenic cotton breeding test field, and continuously and repeatedly performing the kanamycin sulfate identification in the step (3) in the seedling stage. In the full-bloom stage, according to the character complementation principle, using local cotton variety as female parent and using exogenous resource as male parent to prepare hybrid combination, continuously identifying and screening 4 generations, and making the screened F meeting the breeding goal (growth period is less than or equal to 125 days, yield of 5 m row long-stalked cotton is greater than or equal to 0.9kg, average length of upper half portion of fibre is greater than or equal to 29.0mm, breaking specific strength is greater than or equal to 30.0cN/tex and micronaire value is 3.5-5.0)4The seeds of the generation lines are entrusted with the plant ecological environment safety supervision and inspection test center of the department of agricultural rural areas for detection. For the strains qualified in the detection, the next year (F)5Generations) to carry out strain comparison test and survey and identify fusarium wilt and verticillium wilt. Selecting good F5The generation strain (growth period is less than or equal to 125 days, the yield of the ginned cotton is more than or equal to 7 percent compared with the yield of a control variety in Xinjiang (Liaoning) region test, the wilt disease index is less than or equal to 10, the greensickness index is less than or equal to 20, the average length of the upper half part of the fiber is more than or equal to 29.0mm, the breaking ratio strength is more than or equal to 30.0cN/tex, the micronaire value is 3.5-5.0), and the next year (F is less than or equal to 125 days, the yield of the ginned cotton is more than or equal to 7 percent compared with the yield of a control variety in Xinjiang (Liaoning) region test6Generation strain) to carry out different-place multipoint sample ratio test, selecting strains with excellent comprehensive properties to enter the legal test, and obtaining a new non-transgenic cotton variety.
Example Breeding of Liaomian cotton 34, a new variety of very early maturing, high quality, disease resistant and high yield cotton
The tall cotton 34 plant tower type of the new variety of the super-early-maturing, high-quality, disease-resistant and high-yield cotton has the plant height of about 80.8cm, the first fruit branch implantation part is 5-6 nodes, and the implantation height is about 29.4 cm. The average boll of each plant is 10.2, the boll weight is 6.4g, and the clothes score is 41.2%. Growth period is about 124 d. The blooming rate is 95.0%. The fiber main body length is 30.1mm, the regularity index is 83.7 percent, the specific strength is 31.3cN/tex, the micronaire value is 4.6, and the elongation is 6.3 percent. The fusarium wilt refers to 4.4, and the fusarium wilt is highly resistant; verticillium wilt refers to 19.4, and the verticillium wilt is resisted.
In 2014-2015, the cotton seeds are subjected to regional test of the cotton variety in Liaoning province, the yield is increased 11 times, the yield is reduced 1 time, and the yield increasing rate is 91.7%. The average yield per hectare of seed cotton and ginned cotton in two years is 4120.4kg and 1687.8kg respectively, and the yield is increased by 13.7 percent and 14.1 percent respectively compared with the 19 th number of the Liaomiao cotton; the pre-frost bloom rate is 95.0 percent; the yield of the lint cotton before frost per hectare is 1610.1kg, which is increased by 14.7 percent compared with the control; the yield is increased obviously. Production tests are carried out in 2016, the average yield of seed cotton and ginned cotton per hectare is 2988.1kg and 1211.0kg respectively, and the yield is increased by 6.1 percent and 6.1 percent respectively compared with the yield of 19 Liaomian cotton; the blooming rate is 96.2%; the yield of the lint cotton before frost per hectare is 1164.6kg, the yield is increased by 5.7 percent compared with the control, and the yield point rate is 80 percent.
The variety has the characteristics of early maturity, high quality, high yield, stable yield, disease resistance, suitability for mechanical harvesting and the like. Examined by the crop variety examination committee of Liaoning province in 2017, and named as Liao cotton 34 (examined number: Liao examined cotton 2017001).
The super-early-maturing high-quality disease-resistant high-yield cotton new variety Liaomian 34 is bred according to the following steps:
1. in 2006, a gold-labeled Bt-CryIAb/Ac test strip (with the detection precision of 0.125%) is used for detecting 97-3155 (selected line of Xinluaozao No. 17) of a non-transgenic local cotton variety Liaomian No. 16 and Xinjiang. Pulverizing the seeds into powder by a pulverizer, diluting the seeds with distilled water at a ratio of 1:20, inserting the test paper strip into a liquid cup, and obtaining non-transgenic seeds without detection lines of the seeds of No. 16 Liaomiao and No. 97-3155 (namely the selection line of No. 17 Xinluaozao).
2. Single-construction non-transgenic cotton breeding experimental field
Setting an isolation zone (the isolation zone of the unit also comprises trees and houses) of 100-200m with the transgenic cotton test field, independently establishing a non-transgenic cotton breeding test field, and planting the selected seeds of No. 16 Liaomiao cotton and No. 17 Xinluaozao cotton in the non-transgenic cotton breeding test field.
3. Identification of kanamycin sulfate in seedling stage
And (3) spraying kanamycin sulfate aqueous solution with the concentration of 3000mg/kg in a high-temperature sunny day when the cotton seedlings grow 2-4 true leaves, and investigating after 7-10 days. The cotton seedling with the yellow color of the true leaf and the cotyledon is a non-transgenic cotton seedling; the cotton seedling which does not turn yellow (namely is still green) is the Bt gene transfer insect-resistant cotton, and the cotton seedling is required to be completely pulled out. Then, the rest yellow (non-transgenic) cotton seedlings are subjected to final singling (namely, 1 big seedling and strong seedling is reserved in each hole). The retained cotton seedlings continue to grow and develop, blossom and bear bolls, and seed cotton is harvested in autumn.
4. National center of qualification testing
In 10 months, harvested non-transgenic Liaomiao cotton No. 16 and New Luzao No. 17 seed cotton are subjected to cotton ginning to obtain seeds, and 500 g of the seeds of the two varieties (lines) are respectively subjected to detection by a transgenic plant environmental safety supervision and inspection test center of Ministry of agriculture (namely the current plant ecological environmental safety supervision and inspection test center of Ministry of agriculture), so that a detection report of 'no detection of a transgenic Bt gene' is obtained.
5. Crossbreeding
In 2007, non-transgenic Liaoniang cotton No. 16 and New Luzao No. 17 selected seeds which are qualified through detection of 'environmental safety supervision and inspection test center for transgenic plants of Ministry of agriculture' are respectively planted in a non-transgenic cotton breeding test field, and kanamycin sulfate identification in the step 3 is continuously and repeatedly carried out in the seedling stage. In the full-bloom stage (i.e. 19-24 days in 7 months), the cotton variety Liaomian No. 16 with extra early maturity, disease resistance, high yield and low temperature resistance is used as a female parent, the high-quality and high-clothes-division Xinluohao No. 17 is selected as a male parent, a hybridization combination is prepared, namely, sunny weather is selected, at 2-4 afternoon of the day, the stamens of the buds of the female parent are stripped off, and the stigmas (i.e. pistils) are reserved; and picking the flowers of the male parent at 9-11 am on the next day, and smearing the pollen of the stamens of the flowers of the male parent on the stigmas of the flowers of the female parent. F is obtained after successful pollination and development of the resultant cotton boll0Generation hybrid bells. 2008, will F0Planting the generation hybrid bolls in a non-transgenic cotton breeding test field to obtain F1Selecting raw material by selfing combinationThe breeding period is less than or equal to 125 days, the yield of the 5 m long seed cotton is more than or equal to 0.9kg, the average length of the upper half part of the fiber is more than or equal to 29.0mm, the breaking specific strength is more than or equal to 30.0cN/tex, and the micronaire value is 3.5-5.01And (4) generation combination. Good F to be selected in 20091Planting in combination in non-transgenic cotton breeding test field to obtain F2Selfing combination, selecting excellent F in autumn2The generation individual plant (the growth period is less than or equal to 125 days, the average length of the upper half part of the fiber is more than or equal to 29.0mm, the breaking specific strength is more than or equal to 30.0cN/tex, and the micronaire value is 3.5-5.0). In 2010, good F will be selected2Planting the single generation plant in a non-transgenic cotton breeding test field to obtain F3Plant generation and selection of excellent F in autumn3The plant generation row (the growth period is less than or equal to 125 days, the yield of 5 m long-seed cotton is more than or equal to 0.9kg, the average length of the upper half part of the fiber is more than or equal to 29.0mm, the breaking specific strength is more than or equal to 30.0cN/tex, and the micronaire value is 3.5-5.0). In 2011, selected F3The generation plants are planted in a non-transgenic cotton breeding experimental field in rows to obtain F4 generation strains (namely multiple rows of F4Generation), selecting excellent F in autumn4The generation strain (the growth period is less than or equal to 125 days, the yield of 5 m long-row long-staple cotton is more than or equal to 0.9kg, the average length of the upper half part of the fiber is more than or equal to 29.0mm, the breaking specific strength is more than or equal to 30.0cN/tex, and the micronaire value is 3.5-5.0).
The selected Liao 6245 strain seeds of the F4 generation strain, which meet the breeding targets (the growth period is less than or equal to 125 days, the yield of 5 m long-chain cotton is more than or equal to 0.9kg, the down length is more than or equal to 29.0mm, the specific strength is more than or equal to 30.0cN/tex and the micronaire value is 3.5-5.0), are detected by entrusted "inspection and test center for environmental safety supervision of transgenic plants of Ministry of agriculture", and a detection report of 'undetected transgenic Bt gene' is obtained through detection. And (4) passing.
Subjecting the thus obtained F4Seeds of the Liaolian 6245 strain 2012 and 2013 (F)5-F6Generation strain) to carry out strain comparison test and different-place multipoint test, and carrying out investigation and identification on fusarium wilt and verticillium wilt. Obtaining excellent F6The generation strain (growth period 125 days; yield of the ginned cotton is increased by 31.9% compared with 19 Liaomian cotton, wilt disease means 2.3, verticillium wilt means 18.1; average length of the upper half of the fiber is 30.1mm, breaking ratio strength is 31.3cN/tex, and micronaire value is 4.6). 2014 + 2015 Liao6245 participates in the regional test of the cotton variety in Liaoning province, the yield is increased by 11 times, the yield is reduced by 1 time, and the yield increasing ratio is 91.7%.The average yield per hectare of seed cotton and ginned cotton in two years is 4120.4kg and 1687.8kg respectively, and the yield is increased by 13.7 percent and 14.1 percent respectively compared with the 19 th number of the Liaomiao cotton; the pre-frost bloom rate is 95.0 percent; the yield of the lint cotton before frost per hectare is 1610.1kg, which is increased by 14.7 percent compared with the control; the yield is increased obviously. In 2016, the cotton variety production test of Shenliaoning province shows that the average seed cotton and lint cotton yield per hectare is 2988.1kg and 1211.0kg respectively, and the yield is increased by 6.1 percent and 6.1 percent respectively compared with the yield increased by 19 th control Liaoling cotton; the blooming rate is 96.2%; the yield of the lint cotton before frost per hectare is 1164.6kg, the yield is increased by 5.7 percent compared with the control, and the yield point rate is 80 percent.
The variety has the characteristics of early maturity, high quality, high yield, stable yield, disease resistance, suitability for mechanical harvesting and the like. Examined by the crop variety examination committee of Liaoning province in 2017, and named as Liao cotton 34 (examined number: Liao examined cotton 2017001).
Claims (8)
1. A non-transgenic cotton breeding method is characterized in that: crossing the local non-transgenic cotton variety obtained by screening as female parent and the externally introduced non-transgenic cotton variety as male parent in a non-transgenic cotton breeding test field to obtain F0Generation hybrid bells; f is to be0The generation hybrid bolls are continuously selfed for 2 times in a non-transgenic cotton breeding test field, and then F is selected2Inbreeding generation individual plant in non-transgenic cotton breeding test field to obtain F3Planting and transplanting F3The generation plants are planted in a non-transgenic cotton breeding test field for selfing to obtain F4And (4) generating strains, and obtaining the strains of the non-transgenic cotton after identification and screening by using the generation strains.
2. A method of breeding non-transgenic cotton according to claim 1, characterized in that: the female parent and the male parent are screened in such a way that non-transgenic local cotton varieties and externally introduced cotton resource seeds are subjected to non-transgenic detection by a Bt gene transfer test strip, the seeds of the test strip display detection line are cotton seeds containing the Bt gene transfer, and the seeds of the test strip which do not display the detection line are cotton seeds not containing the Bt gene transfer, namely non-transgenic cotton seeds, and are used as a hybrid female parent and a male parent.
3. A method of breeding non-transgenic cotton according to claim 1 or 2, characterized in that: respectively and independently planting the screened female parent seeds and the screened male parent seeds in a non-transgenic cotton breeding test field, spraying kanamycin sulfate aqueous solution in a seedling stage (2-4 true leaves), and keeping cotton seedlings with yellow leaves (namely, non-transgenic cotton seedlings); pulling out the cotton seedling which is not discolored (namely green), keeping the non-transgenic cotton seedling to grow and develop, blooming and bolling, and harvesting the seeds in autumn for later use.
4. A method of breeding non-transgenic cotton according to claim 3, characterized in that: the concentration of the kanamycin sulfate in the kanamycin sulfate aqueous solution is 3000 mg/kg.
5. A method of breeding non-transgenic cotton according to claim 3, characterized in that: non-transgenic cotton seeds obtained after laboratory detection are planted in a non-transgenic cotton breeding test field and are repeatedly identified by an aqueous solution containing kanamycin sulfate in the seedling stage, then larger cotton seedlings with yellow cotyledons or true leaves are reserved, smaller cotton seedlings which are not yellow (namely green) and yellow are pulled out, the reserved larger yellow cotton seedlings grow and develop, blossom and bear bolls, and seeds harvested in autumn are used as breeding parents.
6. A method of breeding non-transgenic cotton according to claim 5, characterized in that: the female parent and the male parent are hybridized after screening, that is, in the full-bloom stage of the cotton (namely, 19-24 days in 7 months), sunny weather is selected, and at 2-4 pm on the day, the stamens of the female parent are stripped off, and the stigma (namely, pistil) is reserved; picking flower of male parent at 9-11 am on the next day, smearing pollen of stamen of flower of male parent on stigmas of flower of female parent, and pollinating cotton boll developed and grown successfully to obtain F0Generation hybrid bells.
7. A method of breeding non-transgenic cotton according to claim 6, characterized in that: the first part is0Planting the generation hybrid bolls in a non-transgenic cotton breeding test field to obtain F1Selfing combination, selecting growth period less than or equal to 125 daysThe yield of 5 m long seed cotton is more than or equal to 0.9kg, the average length of the upper half part of the fiber is more than or equal to 29.0mm, the breaking specific strength is more than or equal to 30.0cN/tex, and the micronaire value is 3.5-5.01Combining the generations; will be excellent F1Planting the generation combination in a non-transgenic cotton breeding test field in the next year to obtain F2Selfing combination, selecting excellent F in autumn2Generation individual plants (growth period is less than or equal to 125 days, average length of the upper half part of the fiber is more than or equal to 29.0mm, breaking ratio strength is more than or equal to 30.0cN/tex, and micronaire value is 3.5-5.0); in the third year, good F will be selected2Planting the single generation plant in a non-transgenic cotton breeding test field to obtain F3Plant generation and selection of excellent F in autumn3The plant generation row (the growth period is less than or equal to 125 days, the yield of the seed cotton growing in 5 m row is more than or equal to 0.9kg, the average length of the upper half part of the fiber is more than or equal to 29.0mm, the breaking specific strength is more than or equal to 30.0cN/tex, and the micronaire value is 3.5-5.0); in the fourth year, F is selected3Planting the plant generation row in a non-transgenic cotton breeding test field to obtain F4Generation line (i.e. multirow F)4Generation), selecting excellent F in autumn4The generation strain (the growth period is less than or equal to 125 days, the yield of the seed cotton growing in 5 m rows is more than or equal to 0.9kg, the average length of the upper half part of the fiber is more than or equal to 29.0mm, the breaking specific strength is more than or equal to 30.0cN/tex, and the micronaire value is 3.5-5.0).
8. A method of breeding non-transgenic cotton according to claim 2, characterized in that: the local cotton varieties are Liaoling cotton No. 18, Liaoling cotton No. 25 and Liaoling cotton No. 16; the cotton resource introduced from the outside includes New Luzao No. 10, New Luzao No. 17, New Luzao No. 61 and New Luzao No. 62.
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