CN1404721A - Semi-matching breeding method for cotton - Google Patents
Semi-matching breeding method for cotton Download PDFInfo
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- CN1404721A CN1404721A CN 02131121 CN02131121A CN1404721A CN 1404721 A CN1404721 A CN 1404721A CN 02131121 CN02131121 CN 02131121 CN 02131121 A CN02131121 A CN 02131121A CN 1404721 A CN1404721 A CN 1404721A
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Abstract
The cotton semigamy breeding method utilizes semigamy reproductive characteristics, uses cotton semigamy material VSG as female parent, and uses transgenic insect-resisting cotton low-generation material as male parent to make hybridization to obtain haploid with transgene in sub-generation, after the haploid is undergo the process of chromosome doubling treatment, the transgenic insect-resisting cotton of zygoid can be cutlured. Said method is used for culturing transgenic insect-resisting cotton, the stable isogenic line can be obtained only through 2-3 generation, the breeding process can be greatly shortened. Four insect-resisting cotton stable lines with different transgene are cultivated, and a transgenic insect-resisting cotton new variety Jinmian No.34 also is cultured.
Description
One, affiliated technical field
The present invention relates to a breeding method that grows cotton, specifically relate to the breeding method of transgenic cotton against pests.
Two, background technology
Cotton is the main economic crops of China, because cotton bollworm harm has caused serious economy loss, only 1992-1993 causes 5,000,000,000 yuan of direct economic losses because of the cotton bollworm great outburst, 10,000,000,000 yuan of indirect economic loss since nineteen nineties.At a large amount of chemical pesticides that use, cotton bollworm produces under the chemical sproof situation many chemical pesticides, U.S. Monsanto company utilized agrobacterium-mediated transformation that the Bt gene is imported cotton 312 kinds of Ke's word in 1988, and transformation is to the word cotton variety of Mount Tai again for 1990-1994, and 1996 begin to apply.The at first synthetic Bt gene of the Chinese Academy of Agricultural Sciences's biotechnology center in 1992,1993 by academy of agricultural sciences, Shanxi Province cotton the unit of grade utilize agrobacterium-mediated transformation that the Bt gene is imported the Shanxi cotton No. 7, identify through test for many years, 1998 by Shanxi Province's variety certification, cotton No. 26 of called after Shanxi, first has the transgenic pest-resistant cotton variety of independent intellectual property right to become China, since applying, has obtained huge economic benefit.
Seed selection process from domestic and international transgenic cotton against pests, general foreign gene profit has agrobacterium-mediated transformation, methods such as pollen tube passage method and particle gun penetrate import anti insect gene in the cotton, since gene make effect mutually, the feature of cotton, characteristic and economical character all can produce and separate and variation, need by systematic breeding or hybridization, technology such as backcross is stablized, generally, import cotton to becoming all stable commercialization kind of resistance and yielding ability from foreign gene, need the 7-8 year, the Harris prediction, if the Bt toxin is simultaneously in cotton, carry out on corn and the jowar and use the control noctuidae pests, so may be in the time of (3-9) in generation at 25-75, will be owing to the generation of pest resistance, and it is useless that this transgenic technology is become.Therefore, insect generation resistance is a challenge to transgenic plant genetic engineering.How shortening the seed selection process of transgenic cotton against pests, quicken the stable of transgene cotton, prolong the service time of transgenic cotton against pests, is problem anxious to be solved.
Three, summary of the invention
Purpose of the present invention just provides a kind of breeding method of transgenic cotton against pests, and this breeding method can the fast and stable transgenic cotton against pests, shortens the breeding process of transgenic cotton against pests.
Partly cooperating reproduction (Semigamy) is a kind of special reproduction type between fertilization (fertilization) and apomixis (apomixis), behind the plant pollination, andro gamete nuclear enters egg cell, but do not merge with egg cell, but division independently of one another, form at last and be inlaid with the heterogeneous embryo that derives from male parent and maternal tissue, this mode of reproduction is called half and cooperates reproduction.
After Turcotte and Feaster (1967) found cotton half compounding ingredient, three during the last ten years, only rested on basic theory aspects such as cytogenetics and embryonic development about the research of half compounding ingredient.Because the self progeny of half compounding ingredient or the monoploid that all can produce certain ratio (6%-20%) with the offspring that it does parent's hybridization have also caused cotton genetic breeding expert's attention.Monoploid has important significance for theories and using value in heredity and breeding, after for example the dyed body of monoploid doubled, can obtain duplicate the isozygotying of two cover chromosomes was that because chromosome isozygotys, the offspring just can not produce separation again.
Utilize haploid above-mentioned characteristic, if in crossbreeding the dyeing number of the haploid gamete of the reorganization of the first generation of hybrid or two generations producer or plant through manually or naturally doubling, just can obtain stable zygoid, obtain stability series and needn't carry out the continuous seed selection of many generations again, shortened breeding process greatly.
It is maternal that the present invention selects cotton half compounding ingredient VSG to do, and makes paternal hybrid with the tired Insect Resistant Cotton of commentaries on classics base is low for material, carries out chromosome doubling with having genetically modified monoploid among the F1, cultivated the transgenic cotton against pests of zygoid.
Cotton half compounding ingredient VSG as female parent belongs to sea-island cotton (G barbadense L), plant height 80-100cm, 135-140 days breeding time, blade 3-5 row, incise darker, the color jaundice, the long 10-15cm of stem internode, about the long 15cm of fruit branch internode, plant is loose, is tower, about the heavy 3g of single bell unginned cotton, fibre length 35-37mm, ginning outturn 28%-30%, seed refers to about 10g.Owing to be subjected to the control of dominant gene Se, can occur the monoploid of 6%-20% in its selfing or the filial generation.Cotton half compounding ingredient VSG of the present invention is introduced by Hebei Prov. Academy of Agricultural ﹠. Forest Sciences's Cotton Research Institute.
The present invention at first selects soil of transgenic Bt cotton to hang down for material 95-1 and does male parent and VSG hybridization, owing to be subjected to the control of the contained Se gene of maternal VSG, has produced about 10% the monoploid with trans Bt gene in F1.
Male parent 95-1 belongs to upland cotton (G hirsutum L), is by academy of agricultural sciences, Shanxi Province agrobacterium-mediated transformation that cotton utilizes that the transgenic cotton against pests that external source anti insect gene Bt imports cotton No. 7 acquisitions in Shanxi is low for material.Because the importing of foreign gene produces the interaction of genes effect, physiological metabolism imbalance in the vine growth and development process, the offspring produces separation, and resistance to insects and other proterties all show instability.Plant height 50-100cm, the long 5-10cm of stem internode, the long 3-15cm of fruit branch internode, plant type has tower, cartridge type etc., and blade is not of uniform size, bottle green, 5 decomposite leafs, 110-140 days breeding time, the heavy 3-5g of single bell unginned cotton, fibre length 25-32mm, ginning outturn 30-40%, seed refers to 8-11g, and the resistance to insects strain difference is bigger, needs just can stablize through successive selection cultivation for many years.
And then we select low the hanging down with the CPTI transgenic Insect Resistant Cotton for material 95-1 of soil of transgenic Bt cotton to carry out biparental cross for material 9701, produced and had the genetically modified first generation of hybrid of Bt+CPTI, use the first generation of hybrid as male parent again, hybridize with half compounding ingredient VSG, the two genetically modified monoploid of valency of Bt+CPTI in F1, occurred having.
The CPTI gene is the cowpea trypsase gene, is a kind of Profilin enzyme gene, and it can destroy the digestive system nerve of insect, thereby causes the insect feeding habits to descend and death.The CPTI gene at first by in the academy of agricultural sciences biotechnology research resultant, academy of agricultural sciences, Shanxi Province cotton institute imports Shanxi cotton No. 12 with it, it is low for material 9701 to have obtained the CPTI transgenic Insect Resistant Cotton.9701 plant height 80-100cm, in 130-135 days vegetative period, plant type is tubular, and blade is big, bottle green, 5 decomposite leafs, the long 5-10cm of stem internode is about the long 10cm of fruit branch internode, about the heavy 5g of single bell unginned cotton, fibre length 29-30mm, ginning outturn 38%-40%, seed refers to about 10g.Growth potential is strong, anti-blight and cotton bollworm.
After obtaining having genetically modified monoploid, need it is cultivated to carry out chromosome doubling.In haploid cultivating process,, improve the condition of growing because therefore the general poor growth, thin and weak all of monoploid will strengthen water and fertilizer management in process of growth.In order to enlarge haploid colony and to improve haploid nutrition condition, the present invention has adopted the method for grafting, is that the parent is hybridized with two different cotton varieties, plants the dwarf with first-filial generation and makes stock, carries out grafting with haploid young shoot do scion.Because first-filial generation has tangible hybrid vigour, moisture and nutriment are abundant, and physiological metabolism is also more vigorous, and the monoploid after the grafting can obtain than moisture and nutriment more sufficient on the original monoploid maternal plant, robust growth helps next step chromosome doubling.
As two parents of monoploid graft stock first-filial generation, can select two non-Insect Resistant Cotton kinds, also can select one or two all is the Insect Resistant Cotton kind, even can hanging down for material as the parent Insect Resistant Cotton.For example the present invention select non-Insect Resistant Cotton kind Shanxi cotton No. 21 with transgenic cotton against pests low for material GR-2 as the parent, the first-filial generation plant that hybridization produces is as stock grafting monoploid young shoot.
The processing method of chromosome doubling has two kinds: seedling doubles to double with branch, and it is under 23-26 ℃ environmental temperature that seedling doubles, and soaks 6 hours in the colchicine solution with the top immersion 0.03-0.05% of monoploid seedling.It then is that growing point with monoploid children shoot immersed in the mixed solution of being made up of 0.03-0.05% colchicin and 5% dimethyl sulfoxide (DMSO) immersion 12-24 hour that branch doubles, and environmental temperature should remain on 28-30 ℃ during immersion.
After the dyed body of monoploid doubles, become zygoid.To zygoid carry out that resistance to insects is measured, quality is identified and the output comparative test after, stable the isozygotying that promptly becomes a transgenic cotton against pests is.
The present invention adopts semi-matching breeding method seed selection transgenic cotton against pests, only need 2-3 for the stability series that just can obtain transgenic cotton against pests, and general transgenic cotton against pests since gene make effect mutually, need by systematic breeding or hybridization, technology such as backcross is stablized, and needs continuous seed selection 5-7 for just obtaining stability series.Therefore, utilize semi-matching breeding method to shorten breeding process greatly, improved breeding efficiency.
Because the gene pure of the transgenic cotton against pests stability series that the employing semi-matching breeding method obtains, difficult generation makes a variation and degenerates, and stability is better than general kind, and the time of production application is also longer.
Four, specific embodiment
With half compounding ingredient VSG is female parent, and low with transgenic cotton against pests is that male parent is hybridized for material, obtains haplobiont 337 strains.98 strains of (Bt+CPTI) bivalent gene monoploid are changeed in wherein trans Bt gene monoploid 239 strains.After dyed body doubles, obtain 49 strains of trans Bt gene doubled haploid altogether, change 21 strains of (Bt+CPTI) bivalent gene doubled haploid.
Identify by field plant, strain system product ratio, from breeding time, plant height, single bell unginned cotton weight, ginning outturn, seed refer to, aspect such as lint yield investigates analysis comprehensively, and by the evaluation of solarium's resistance to insects with through cotton quality supervision and inspection center of Ministry of Agriculture cotton fibre quality mensuration, from 49 trans Bt gene doubled haploid strain systems, filter out three stability series DHR-1, DHR-2, DHR-3, from the two valency doubled haploid of 21 commentaries on classics (Bt+CPTI) strain system, filter out two stability series DHR-4, DHR-5.Wherein DHR-2 is after further by regional test and production test, and in February, 2002, cotton No. 34 of called after Shanxi became the transgenic cotton against pests new varieties by the authorization of the Shanxi Province variety certification committee.
Four stability series features of table 1 and cotton fibre quality
| ?DHR-1 | ?DHR-3 | ?DHR-4 | ?DHR-5 | |
| Breeding time (my god) the single bell seed cotton heavy of plant height (cm) (g) seed refers to (g) ginning outturn (%) fibre length (mm) regularity (%) specific strength (CN/tex) percentage elongation (%) mic value gas yarn quality | ?116 ?64.7 ?4.8 ?10.1 ?40.1 ?30.6 ?43.9 ?20.0 ?6.6 ?3.7 ?1981 | ?125 ?74.3 ?5.3 ?10.4 ?38.3 ?31.8 ?42.2 ?21.8 ?6.4 ?4.1 ?2065 | ?128 ?80.2 ?5.1 ?11.7 ?39.2 ?31.7 ?45.4 ?20.8 ?7.7 ?3.2 ?2087 | ?131 ?81.1 ?5.3 ?12.2 ?38.7 ?31.2 ?41.7 ?21.9 ?6.7 ?3.2 ?2146 |
Four stability series lint yields of table 2
| Lint yield (kg/ mu) | Than CK1 volume increase (%) | Than CK2 volume increase (%) | |
| The pest-resistant contrast of DHR-1 DHR-3 DHR-4 DHR-5 95-1, (CK1) non-pest-resistant contrast Shanxi is cotton No. 10, (CK2) | ?63.7 ?63.4 ?71.3 ?72.4 ?61.2 ?55.4 | ?4.1 ?3.6 ?16.5 ?18.3 | ?15.0 ?14.4 ?28.7 ?30.7 |
Four pest-resistant qualification results in stability series solarium of table 3
| Hundred strain larvas (head) | Reduce (%) than CK | The killed flower bud of hundred strains (individual) | Reduce (%) than CK | |
| Cotton No. 10 (CK) in DHR-1 DHR-3 DHR-4 DHR-5 contrast Shanxi | ????11 ????12 ????11 ????8 ????63 | ????82.5 ????80.9 ????82.5 ????87.3 | ????26 ????29 ????32 ????19 ????267 | ??90.3 ??89.1 ??88.0 ??92.8 |
In the above-mentioned variety comparative test, contrast 95-1 is that soil of transgenic Bt cotton is low for material, and the Shanxi is a non-transgenic Insect Resistant Cotton kind cotton No. 10.In four stability series, DHR-1, DHR-3, DHR-4, resistance to insects and 95-1 are more or less the same, and the resistance to insects of DHR-5 is a little more than 95-1.
DHR-2 carries out variety comparative test at the soil of transgenic Bt cotton stability series, under insect receiving net chamber isolation condition, do not control cotton bollworm the time of infertility, two annual lint yield 58.8kg/ mus, comparison under the open cultivation condition of routine, prevents and treats cotton bollworm according to Shanxi cotton No. 21 (non-transgenic Insect Resistant Cotton) volume increase 53.6% time of infertility, two annual lint yield 61.3kg/ mus, comparison is according to cotton No. 21 volume increase 8.3% in Shanxi.2000 five somes participation production tests, 0.5 mu of sub-district area, five points all increase production, average lint yield 76.5kg/ mu, comparison is according to cotton No. 21 volume increase 12.8% in Shanxi, and calendar year 2001 expands eight points to and participates in production test, average lint yield 88.7kg/ mu, eight points all show volume increase, and comparison is according to cotton No. 21 volume increase 29.2% in Shanxi.Through the field evaluation, DHR-2 growth potential is strong, and growth is neat, and boll per plant is many and sane, and cotton boll is big and even, and resistance to insects is strong.DHR-2 plant type compactness, plant height 70-80cm, 119 days breeding times, about the heavy 5g of single bell unginned cotton, seed refers to 10.2g, ginning outturn 38-40%, dark green leaf color, medium big, growth potential is stronger, later stage leaf wadding is concentrated, and the flower rate is more than 80% before the frost, and the fiber knot is white, measure through Ministry of Agriculture cotton quality verification test center, 2.5% span length 30.3mm, regularity 47.4%, specific strength 21.1CN/tex, tensile stretch 9.7%, be worth 402 with the clone, reflectivity 79.4%, yellow degree 8.6, ring thread yarn is strong by 129, gas yarn quality 1895 reaches national 95 breedings tackling key problem index, conforms to the textile industry requirement.
Identify through the solarium, DHR-2 2 generation 15 of cotton bollworm hundred strain larvas, 63 of the cotton No. 10 hundred strain larvas in contrast Shanxi, comparison is according to reducing 76.2%, 41 of the killed flower buds of DHR-2 hundred strains, 267 of the cotton No. 10 hundred killed flower buds of strain in contrast Shanxi, comparison has reached high anti-level according to reducing 84.6%.2000 and 200 years are in 2 generations during the cotton bollworm occurrence in peak period, respectively 13 points are produced investigation, 11.1 of DHR-2 2 generation cotton bollworm hundred strain larvas two annuals, the contrast Shanxi is 40.2 cotton No. 21, the DHR-2 comparison is 45.9 according to reducing killed flower bud DHR-2 two annuals of 71.3%, hundred strain, and the contrast Shanxi is 240 cotton No. 21, the DHR-2 comparison reaches high anti-level according to reducing 72.4%.DHR-2 authorized by the Shanxi Province variety certification committee in February, 2002, cotton No. 34 of called after Shanxi.
Claims (10)
1, a kind of semi-matching breeding method for cotton, it is characterized in that making parent with cotton half compounding ingredient VSG, carry out chromosome doubling with having genetically modified monoploid in the first filial generation with transgenic cotton against pests is low again for the material paternal hybrid, cultivate the transgenic cotton against pests of zygoid.
2, semi-matching breeding method for cotton according to claim 1, it is characterized in that making parent with cotton half compounding ingredient VSG, hybridize as male parent for material 95-1 so that soil of transgenic Bt cotton is low, obtain trans Bt gene monoploid, after the chromosome doubling, cultivate the soil of transgenic Bt cotton of zygoid.
3, semi-matching breeding method for cotton according to claim 1, it is characterized in that doing maternal with cotton half compounding ingredient VSG, hybridize as male parent for the material 95-1 and the low first generation of hybrid of CPTI transgenic Insect Resistant Cotton so that soil of transgenic Bt cotton is low for material 9701 hybridization, obtain changeing (Bt+CPTI) gene monoploid, after the chromosome doubling, cultivate commentaries on classics (Bt+CPTI) the gene pest-resistant cotton of zygoid.
4, semi-matching breeding method for cotton according to claim 1 is characterized in that with two different cotton varieties being that the parent is hybridized, and makes stock with the first-filial generation plant, and haploid cultivation is carried out in monoploid sprout grafting.
5, semi-matching breeding method for cotton according to claim 1 is characterized in that in two cotton varieties as the parent, and it all is that Insect Resistant Cotton kind or Insect Resistant Cotton are low for material that a kind or two kinds are arranged.
6, semi-matching breeding method for cotton according to claim 1 is characterized in that carrying out chromosome doubling with in the top immersion of the monoploid seedling 0.03%-0.05% colchicine solution 6 hours.
7, semi-matching breeding method for cotton according to claim 1 is characterized in that growing point with monoploid children shoot immersed in the mixed solution of 0.03%-0.05% colchicin and 5% dimethyl sulfoxide (DMSO) 12-24 hour, carried out chromosome doubling.
8, the Insect Resistant Cotton kind of utilizing claim 1 or 2 breeding methods to obtain is characterized in that half compounding ingredient VSG and soil of transgenic Bt cotton are hanged down the hybridization for material 95-1, obtains transgenic pest-resistant cotton variety Shanxi after the dyed body of first filial generation monoploid doubles cotton No. 34.
9, the Insect Resistant Cotton kind of utilizing claim 1 or 2 breeding methods to obtain, it is characterized in that half compounding ingredient VSG and soil of transgenic Bt cotton are hanged down the hybridization for material 95-1, obtain transgenic cotton against pests stability series DHR-1, DHR-3 after the dyed body of first filial generation monoploid doubles.
10, the Insect Resistant Cotton kind of utilizing claim 1 or 3 breeding methods to obtain, it is characterized in that half compounding ingredient VSG and low the hanging down for the first generation of hybrid of material 9701 hybridization with the CPTI transgenic Insect Resistant Cotton for material 95-1 of soil of transgenic Bt cotton are hybridized, the dyed body of first filial generation monoploid add amass wealth by heavy taxation after, obtain transgenic cotton against pests stability series DHR-4, DHR-5.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116210582A (en) * | 2023-04-13 | 2023-06-06 | 南阳市农业科学院(南阳市科学院) | Breeding method for rapidly stabilizing distant hybridization offspring of cotton |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116210582A (en) * | 2023-04-13 | 2023-06-06 | 南阳市农业科学院(南阳市科学院) | Breeding method for rapidly stabilizing distant hybridization offspring of cotton |
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