CN103828708A - Efficient soybean cytoplasmic male sterility test-crossing method and device - Google Patents
Efficient soybean cytoplasmic male sterility test-crossing method and device Download PDFInfo
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- CN103828708A CN103828708A CN201410097321.8A CN201410097321A CN103828708A CN 103828708 A CN103828708 A CN 103828708A CN 201410097321 A CN201410097321 A CN 201410097321A CN 103828708 A CN103828708 A CN 103828708A
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Abstract
The invention provides an efficient soybean cytoplasmic male sterility test-crossing method and device, and relates to the determination on soybean cytoplasmic male sterility maintainer lines, restoring lines and restoring capability. The method comprises the following steps: breeding sterile lines and restoring lines, wherein three sterile lines, which can be restored to different extents by the restoring lines, are used as test-crossing female parents, and materials to be tested are used as male parents; dibbling the test-crossing female parents and the male parents to be tested in groups in a field, independently covering each test-crossing unit with a detachable test-crossing mesh chamber 3 to 5 days before the blooming of the materials to be tested, putting a certain number of megachile rotundata fabricius or artificially domesticated bees into each test-crossing mesh chamber for the pollination of soybeans in the full-blossom period of the test-crossing male and female parents, removing the test-crossing mesh chambers after the blooming period, and harvesting F1 hybrid pods according to combinations after ripening; and identifying the pollen abortion rate of F1 plants in the next planting season, determining a restoring and maintaining relationship, and determining the restoring type of the F1 plants if the F1 plants belong to restoring lines. The method and the device are suitable for the large-scale determination on large quantities of germplasm; artificial crossing is replaced by insect pollination, so that time and labor are saved, and the efficiency is high; different types of sterile lines are used as the test-crossing female parents, and whether the materials to be tested belong to maintainer lines or restoring lines and the restoring types of the materials can be tested by one test crossing.
Description
technical field:
Invention provides a kind of male sterile test cross method of soya cells matter and device efficiently, relate to the mensuration of mensuration, restorer and the recovery capability of soya cells matter male sterility maintainer line, profit is seed selection male sterile line and restorer in this way, belongs to crop hybrid kind breeding technique field.
background technology:
" three are " crossbreed is the F being obtained by male sterile line and restorer hybridization
1seed." three are " comprises male sterile line, maintainer and restorer, female the educating of the male sterile of male sterile line own, and can only accept external pollen could be solid; Maintainer has the sterile characteristic of the male sterile line of maintenance, to male sterile line pollination, can continue to obtain male sterile line; Restorer has makes male sterile line recover the characteristic that normally can educate, normal to bear seeds fertility restorer in the time that the next generation plants after male sterile line pollination, is crossbreed.In the seed selection of crossbreed, male sterile line and restorer are basic materials.Therefore first arbitrary germ plasm resource need to determine its extensive guarantor's relation, can transformation be male sterile line if maintainer backcrossed through too much generation, if restorer, can in breeding, directly utilize or as breeding intermediate materials indirect utilization according to characteristics such as its recovery extent, coordinate force.Conventional test cross method is mainly to utilize single male sterile line as test cross female parent at present, and detected materials obtains F as male parent artificial pollination hybridization
1hybridization pod, and at next plantation season plantation F
1seed, identifies F
1the pollen abortion rate of plant is determined its extensive guarantor's relation.
Conventional test cross method there is following shortcoming: the one, artificial hybridization survival rate is not high, efficiency is low, is not suitable in extensive test cross, particularly breeding the test cross poor operability to a large amount of improved progeny colony; The 2nd, there is considerable male sterile line, can be recovered by some male sterile line, but can not be recovered by other restorers, even if can be recovered by some male sterile line, but do not reflect the recovery capability of restorer.Utilize single maternal test cross, easily miss the restorer that can only recover some particular type male sterile line, cannot determine its recovery capability and degree accurately simultaneously.Therefore, conventional test cross method can not meet germplasm test cross needs in enormous quantities completely.
summary of the invention:
The invention provides a kind of male sterile test cross method of soya cells matter efficiently, solve the shortcomings of existing test cross method, have save time, saving of labor, method are simple, easy to operate, reliable results, efficiency advantages of higher.
The present invention also provides the device that is applicable to said method.
the male sterile test cross method of the efficient soya cells matter of one provided by the invention, is characterized in that:
Utilize simultaneously one group typical, dissimilar sterile be test cross female parent, under isolation condition, adopt the many female collocation methods of a father, utilize insect pollination, it is maintainer or restorer and different recovery types that a test cross can be measured detected materials.
the male sterile test cross method of the efficient soya cells matter of one disclosed by the invention, comprises the following steps:
1) select the maternal male sterile line of test cross:
The first test cross female parent, can be kept by all maintainers, and such male sterile line can be recovered by all restorers simultaneously, F
1pollen abortion rate, between 0-10%, belongs to and is very easily resumed type, take JLCMS82A and JLCMS101A as representative, is also included in similar other male sterile lines in the degree of being resumed;
The second test cross female parent, can be kept by all maintainers, can be exceeded 50% restorer recovery simultaneously, F
1pollen abortion rate, between 11-60%, belongs to and is easily resumed type, take JLCMS9A as representative, is also included in similar other male sterile lines in the degree of being resumed.This class male sterile line can be exceeded 50% restorer recovery;
The 3rd test cross female parent, can be kept by all maintainers, only can be less than 10% restorer simultaneously and recover, F
1pollen abortion rate is greater than 61%, belongs to difficulty and is resumed type, take JLCMS89A and JLCMS31A as representative, is also included in similar other male sterile lines in the degree of being resumed;
2) solarium for test cross:
Test cross is to form enclosure space by reinforcing bar square frame skeleton and the gauze cover that covers on it with solarium, and test cross is covered with zipper opening with the side gauze of solarium;
3) field sowing mode:
Sowing season, in land for growing field crops, by community sowing, three kinds of test crosses of step 1) are maternal forms a test cross community with 1 male parent to be measured, each community sowing 1-2 cave male parent to be measured and three kinds, 4-5 cave test cross female parent; Cell size be vertical ridge to long 80-130cm, parallel ridge is to wide 80-120cm; Seeding method is bunch planting, and the first ridge planting order is the first test cross female parent, male parent to be measured and the 3rd test cross female parent, and the second ridge planting order is the second test cross female parent, male parent to be measured and the 3rd test cross female parent, and seeding quantity is 4, every cave, final singling 2 strains after emerging; According to suitably adjusting father, maternal sowing time in father, maternal flowering stage, consistent to guarantee father, maternal flowering stage; When the recovery capability of test cross restorer, can select simultaneously male sterile line sowing that 1-2 coordinate force is high in community with a small amount of hybrid seed of restorer hybridization preparation;
This planting patterns only represents a male parent material of point bunch planting kind and many parts of maternal materials in test cross solarium, and the sowing order of actual field operation and cave are apart from meeting under material energy normal growth condition and can suitably adjusting; " ridge " is in flat planting Qu Ye representative " OK ";
4) set up solarium for test cross:
Within 3-5 days before test cross father, female parent are bloomed, give on each test cross community and set up a step 2) solarium for test cross, and remove wild insect in solarium;
5) the maternal pollen abortion rate of test cross checks:
Before button net, give each test cross father, maternal listing mark, after button net, with the maternal male sterile line pollen fertility of sediments microscope inspection test cross, pull out pollen abortion rate lower than 98% plant;
6) insect pollinator is thrown in:
At test cross father, maternal full-bloom stage, each test cross is thrown in 2-3 clover megachilid to the maternal pollination of test cross with solarium, for guaranteeing pollination quality, meets rainy weather and can repeat to throw in clover megachilid one time; If with honey bees pollination, adopt miniature little case bee colony, 100 left and right of bee colony size;
7) insect pollinator management:
Clover cuts after Ye Feng throws in does not need extra labor management, and the honeybee syrup of need to regularly feeding, for drink clear water;
8) hybridization pod results:
On test cross maternal plant, hybridize after pod maturation, also record in detail seed source by combination and numbering results hybridization pod;
9) hybridization F
1plant pollen abortion rate checks: next plantation season, and by combined grow hybridization F
1seed, by observing F
1plant pollen abortion rate, determines extensive guarantor's relation of male parent material to be measured, if restorer is determined the type of restorer.
the male sterile test cross method of efficient soya cells matter of the present invention, is characterized in that the field planting mode that step 3) relates to is:
Sowing season, field is by community sowing, and three kinds of test cross female parents form a test cross community with 1 male parent to be measured, each community sowing 1-2 cave male parent to be measured and three kinds, 4-5 cave test cross female parent; Cell size be vertical ridge to long 80-130cm, parallel ridge is to wide 80-120cm; Seeding method is bunch planting, and the first ridge planting order is the first test cross female parent, male parent to be measured and the 3rd test cross female parent, and the second ridge planting order is the second test cross female parent, male parent to be measured and the 3rd test cross female parent, and seeding quantity is 4, every cave, final singling 2 strains after emerging; According to suitably adjusting father, maternal sowing time in father, maternal flowering stage, consistent to guarantee father, maternal flowering stage; When the recovery capability of test cross restorer, can select 1-2 male sterile line sowing a small amount of hybrid seed of restorer hybridization preparation to be measured with it in community that coordinate force is high simultaneously.
This planting patterns only represents divides a male parent material of bunch planting kind and many parts of maternal materials a test cross solarium, in the practical operation of field, sowing order and cave are apart from can suitably adjusting according to environmental condition and tree characteristics.
it is as follows that the present invention has remarkable result compared with conventional test cross method:
(1) test cross information is full and accurate, by a test cross, can obtain detected materials correlated inheritance information comprehensively and accurately.
(2) pollination efficiency is high, and with megachilid or honey bees pollination, high outcrossing rate male sterile line is the highest gathers in the crops more than 150 hybrid seed, and low outcrossing rate male sterile line also can be gathered in the crops more than 20 hybrid seed.
(3) method of operating is simple, without extra field management measure, can directly on ridge, field, carry out, before blooming and the field management of florescence after finishing identical with production field.
(4) be applicable to the extensive test cross in field, this technology has solved the artificial Crossing system of very difficult use and a large amount of germplasms has been carried out to test cross and obtain enough technical problems of hybridization pod, the particularly test cross of a large amount of germplasms or filial generation colony.
accompanying drawing explanation:
Fig. 1 is test cross of the present invention solarium's structural representation;
Wherein, 1, skeleton; 2, cover net; 3, zipper opening;
Fig. 2 is test cross community, field of the present invention sowing schematic diagram;
Wherein, ♀ 1A: the maternal male sterile line JLCMS82A of the first test cross or JLCMS101A; ♀ 2A: the maternal male sterile line JLCMS9A of the second test cross; ♀ 3A: the maternal male sterile line JLCMS89A of the 3rd test cross or JLCMS31A; ♂: arbitrary male parent soybean germplasm to be detected;
Fig. 3 is the present invention's front field test cross solarium's scheme of installation of blooming.
embodiment:
embodiment 1:
1. select tester:
Select the male sterile line of three types as test cross female parent: (1) is very easily resumed male sterile line: JLCMS82A; (2) male sterile line being easily resumed: JLCMS9A; (3) male sterile line that difficulty is resumed: JLCMS89A.
Select arbitrary germplasm to be measured as male parent to be measured.
2. prepare solarium for test cross:
The reinforcing bar that is 10mm with diameter welding solarium for test cross: be Simple assembled demountable structure, the length of skeleton 1 is respectively 80-130cm × 80-120cm × 130-160cm, customization gauze: gauze cover 2 specifications are 20 orders, on skeleton 1, gauze cover 2 sizes and shape and skeleton 1 match, so that install, the zipper opening 3 that there is high 100-150cm gauze cover 2 sides, refers to Fig. 1.
3. field sowing:
Sowing season, by community, at field sowing, three test cross female parents form a test cross community with 1 male parent to be measured, each community sowing 1-2 cave male parent to be measured and three kinds, 4-5 cave test cross female parent; Cell size be vertical ridge to long 80-130cm, parallel ridge is to wide 80-120cm; Seeding method is bunch planting, and the first ridge planting order is JLCMS82A, male parent to be measured and JLCMS89A, and the second ridge planting order is JLCMS9A, male parent to be measured and JLCMS89A, and seeding quantity is 4, every cave, final singling 2 strains after emerging; According to suitably adjusting father, maternal sowing time in father, maternal flowering stage, consistent to guarantee father, maternal flowering stage.
4. set up solarium for test cross:
Within 3-5 days before test cross father, female parent are bloomed, give on each test cross community and set up a step 2) solarium for test cross, refer to Fig. 3, remove all insects in solarium with broad spectrum, low residue insecticide.
5. check the maternal pollen abortion rate of test cross:
Before button net, the father to each test cross community, maternal listing mark; After button net, come into bloom at test cross female parent, by its pollen abortion rate of microexamination, extract pollen abortion rate lower than 98% plant, purity as maternal in test cross is high, also can hand inspection male sterile line pollen.
6. throw in clover megachilid:
At the sunny weather of test cross father, female full-bloom stage, 2-3 clover megachilid thrown in to the maternal pollination of test cross by each solarium, for guaranteeing pollination quality, meets rainy weather and can repeat to throw in clover megachilid one time.
7. management clover megachilid:
Throw in after clover megachilid, in 50 meters, test region and periphery thereof, ban use of the virose agricultural chemicals of honeybee class, until pollination finishes, clover cuts after Ye Feng throws in does not need extra labor management.
8. results hybridization pod:
In the time hybridizing pod maturation on test cross maternal plant, results hybridization pod is also recorded hybrid seed source in detail by number.
9. hybridization F
1plant pollen abortion rate checks:
In next plantation season, sow hybridization F in field by combination
1seed, samples and uses microscopic examination F flowering stage
1plant pollen abortion rate, determines extensive guarantor's relation of detected materials, if maintainer, the number generation of continuing to backcross can obtain new male sterile line; If restorer, according to its performance of recovery to dissimilar male sterile line, is determined the type of restorer.
?1. tester selects:
Using the male sterile line of three types as test cross female parent: (1) is very easily resumed male sterile line: JLCMS101A; (2) male sterile line being easily resumed: JLCMS9A; (3) male sterile line that difficulty is resumed: JLCMS31A.
Select arbitrary germplasm to be measured as male parent to be measured.
2. prepare the special solarium of test cross:
The reinforcing bar that is 10mm with diameter welding solarium for test cross: be Simple assembled demountable structure, the length of skeleton 1 is respectively 80-130cm × 80-120cm × 130-160cm, customization gauze: gauze cover 2 specifications are 20 orders, on skeleton 1, gauze cover 2 sizes and shape and skeleton 1 match, so that install, the zipper opening 3 that there is high 100-150cm gauze cover net 2 sides, refers to Fig. 1.
3. field sowing:
Sowing season, by community, at field sowing, 3 test cross female parents form a test cross community with 1 male parent to be measured, each community sowing 1-2 cave male parent to be measured and three kinds, 4-5 cave test cross female parent; Cell size be vertical ridge to long 80-130cm, parallel ridge is to wide 80-120cm; Seeding method is bunch planting, and the first ridge planting order is JLCMS101A, male parent to be measured and JLCMS31A, and the second ridge planting order is JLCMS9A, male parent to be measured and JLCMS31A, and seeding quantity is 4, every cave, final singling 2 strains after emerging; According to suitably adjusting father, maternal sowing time in father, maternal flowering stage, consistent to guarantee father, maternal flowering stage;
4. set up solarium for test cross:
Within 3-5 days before test cross father, female parent are bloomed, give on each test cross community and set up a step 2) solarium for test cross, refer to Fig. 3, remove all insects in solarium with broad spectrum, low residue insecticide.
5. check the maternal pollen abortion rate of test cross:
Before button net, the test cross father to each community, female listing mark; After button net, come into bloom at test cross female parent, by its pollen abortion rate of microexamination, extract pollen abortion rate lower than 98% plant, purity as maternal in test cross is high, also can hand inspection male sterile line pollen.
6. honeybee is thrown in:
At the sunny weather of test cross father, maternal full-bloom stage, 1 little case honey bees pollination is thrown in by each solarium, 100 left and right of bee colony size.
7. honeybee management:
Throw in after honeybee, in 50 meters, test region and periphery thereof, ban use of the virose agricultural chemicals of honeybee, until pollination finishes.The honeybee syrup of need to regularly feeding, for drink clear water.
8. hybridization pod results:
In the time hybridizing pod maturation on test cross maternal plant, results hybridization pod is also recorded hybrid seed source in detail by number.
9. hybridization F
1plant pollen fertility checks:
Next planting season, hybridize F in field by combination sowing
1seed, samples and uses microscopic examination pollen abortion rate flowering stage, determines extensive guarantor's relation of detected materials, if maintainer, the number generation of continuing to backcross can obtain new male sterile line; If restorer, according to its performance of recovery to dissimilar male sterile line, is determined the type of restorer.
1. select the male sterile line of three types as test cross female parent:
(1) be very easily resumed male sterile line: JLCMS82A; (2) male sterile line being easily resumed: JLCMS9A; (3) male sterile line that difficulty is resumed: JLCMS31A.
Select arbitrary germplasm to be measured as male parent to be measured.
2. prepare the special solarium of test cross:
The reinforcing bar that is 10mm with diameter welding solarium for test cross: be Simple assembled demountable structure, the length of skeleton 1 is respectively 80-130cm × 80-120cm × 130-160cm, customization gauze: gauze cover 2 specifications are 20 orders, on skeleton 1, gauze cover 2 sizes and shape and skeleton 1 match, so that install, the slide fastener 3 that there is high 100-150cm gauze cover 2 sides, refers to Fig. 1.
3. field sowing:
Sowing season, by community, at field sowing, three kinds of test cross female parents form a test cross community with 1 male parent to be measured, each community sowing 1-2 cave male parent to be measured and three kinds, 4-5 cave test cross female parent; Cell size be vertical ridge to long 80-130cm, parallel ridge is to wide 80-120cm; Seeding method is bunch planting, and the first ridge planting order is JLCMS82A, male parent to be measured and JLCMS31A, and the second ridge planting order is JLCMS9A, male parent to be measured and JLCMS31A, and seeding quantity is 4, every cave, final singling 2 strains after emerging; According to suitably adjusting father, maternal sowing time in father, maternal flowering stage, consistent to guarantee father, maternal flowering stage.
4. set up solarium for test cross:
Within 3-5 days before test cross father, female parent are bloomed, give on each test cross community and set up a step 2) solarium for test cross, refer to Fig. 3, remove all insects in solarium with broad spectrum, low residue insecticide.
5. check the maternal pollen abortion rate of test cross:
Before button net, the father to each test cross community, maternal listing mark; After button net, come into bloom at test cross female parent, by its pollen abortion rate of sediments microscope inspection, extract pollen abortion rate lower than 98% plant, purity as maternal in test cross is high, also can hand inspection male sterile line pollen.
6. throw in clover megachilid:
At the sunny weather of test cross father, maternal full-bloom stage, 2-3 clover megachilid thrown in to the maternal pollination of test cross by each solarium, for guaranteeing pollination quality, meets rainy weather and can repeat to throw in clover megachilid one time.
7. management clover megachilid:
Throw in after clover megachilid in 50 meters, test region and periphery thereof and ban use of the virose agricultural chemicals of honeybee class, until pollination finishes, clover cuts after Ye Feng throws in does not need extra labor management.
8. hybridization pod results:
In the time hybridizing pod maturation on test cross maternal plant, results hybridization pod is also recorded hybrid seed source in detail by number.
9. hybridization F
1plant pollen fertility checks:
Next year spring, in field by combination sowing hybridization F
1seed, samples and uses microscopic examination pollen abortion rate flowering stage, determines extensive guarantor's relation of detected materials, if maintainer, the number generation of continuing to backcross can obtain new male sterile line; If restorer, according to its performance of recovery to dissimilar male sterile line, is determined the type of restorer.
show the good effect of patent of the present invention by following test:
test example 1:
Checking embodiment 1 measures the recovery type of soybean RN type sterile cytoplasm maintainer and restorer.
(1) optional 3 parts of soybean materials carry out test cross, are respectively JLCMS8B, JLCMSR4 and S37.
(2) field planting and bloom before field management, the sowing of 3 communities is divided in field, community one sowing order: the first ridge is respectively JLCMS82A, JLCMS8B and JLCMS89A, and parallel the second ridge is respectively JLCMS89A, JLCMS8B and JLCMS9A, and cave is apart from 40cm; Vertical ridge is to second and third community of sowing, and adjacent two minizones are every 1 ridge, and male parent is respectively JLCMSR4 and S37, and test cross father, planting of maternal mode are with first community; Before blooming, farming method is with Soybean production field.
(3) before blooming to all test cross fathers, maternal listing mark, simultaneously on test cross community, solarium for test cross is set up in assembling, and all test cross fathers, female parent are covered in solarium.With reference to Fig. 3.
(4) the maternal pollen abortion rate of test cross checks, chooses and is about to open bud at the maternal initial bloom stage of test cross, and 3 flowers are got in every strain, fixing for subsequent use with 70% ethanol; At the indoor I of experiment
2-KI decoration method is observed pollen abortion rate, pulls out pollen abortion rate lower than 98% plant.
(5) clover megachilid is thrown in, manages: at the sunny weather of test cross father, maternal full-bloom stage, 2-3 clover megachilid thrown in to the maternal pollination of test cross by each solarium.Throw in after clover megachilid, in 50 meters, test region and periphery thereof, ban use of the virose agricultural chemicals of clover megachilid, until pollination finishes.Clover cuts after Ye Feng throws in does not need extra labor management.
(6) hybridization pod results: in the time hybridizing pod maturation on test cross maternal plant, by combination and numbering results hybridization pod, JLCMS8B, as the community one of top-cross male parent, gathers in the crops respectively 151,59 and 32 hybrid seeds on JLCMS82A, JLCMS9A and JLCMS89A; JLCMSR4 is as the community two of top-cross male parent, gathers in the crops respectively 128, seed, 47 and 22 on JLCMS82A, JLCMS9A and JLCMS89A; S37 is as the community three of top-cross male parent, gathers in the crops respectively 118, seed, 41 and 20 on JLCMS82A, JLCMS9A and JLCMS89A.
(7) Second Year is pressed the hybridization F of combined grow San Ge community spring
1seed, 15 seeds of each combination sowing, be about to open bud sampling to 10 of each combination individual plants flowering stage, with 75% alcohol fixation, tests indoor I
2-KI decoration method is observed pollen abortion rate, and average pollen abortion rate be the results are shown in to table 1.
3 parts of test cross male parent F of table 1
1
pollen abortion rate statistics (%)
(8) test cross result, three test cross female parents are hybridized F with JLCMS8B as can be seen from Table 1
1pollen abortion rate all to exceed 98.9%(highly sterile), determine that JLCMS8B is maintainer; JLCMSR4 can recover all three class male sterile lines, particularly can recover the male sterile line JLCMS89A that is difficult for being resumed, its F
1pollen abortion rate is 30.7%, can determine that JLCMSR4 is a strong restorer; The maternal hybridization F with S37 of three test crosses
1pollen abortion rate all exceed 60%, but do not reach highly sterile standard, can determine S37 neither restorer neither maintainer.
test example 2
Checking embodiment 2 measures the recovery type of RN type sterile cytoplasm maintainer and restorer.
(1) optional 3 parts of materials carry out test cross, are respectively JLCMS14B, JLCMSR2 and S39.
(2) field planting and bloom before field management, 3 community sowings are divided in field, the first community sowing order: the first row is respectively JLCMS101A, JLCMS14B and JLCMS31A, and parallel the second row is respectively JLCMS31A, JLCMS14B and JLCMS9A, and cave is apart from 40cm; Vertical ridge is to second and third community of sowing, and adjacent two minizones are every 1 ridge, and male parent is respectively JLCMSR2 and S39, and test cross father, planting of maternal mode are with first community; Before blooming, farming method is with Soybean production field.
(3) before blooming to all test cross fathers, maternal listing mark, simultaneously on test cross community, solarium for test cross is set up in assembling, and all test cross fathers, female parent are covered in solarium.With reference to Fig. 3.
(4) the maternal pollen abortion rate of microscopy, chooses and is about to open bud at the maternal initial bloom stage of test cross, and 3 flowers are got in every strain, fixing for subsequent use with 70% ethanol; At the indoor I of experiment
2-KI decoration method is observed pollen abortion rate, pulls out pollen abortion rate lower than 98% plant.
(5) honeybee is thrown in, manages: at the sunny weather of test cross father, maternal full-bloom stage, a little case honey bees pollination is thrown in by each solarium, 100 left and right of bee colony size, throw in after honeybee in 50 meters, test region and periphery thereof and ban use of the virose agricultural chemicals of honeybee, until pollination finishes.The honeybee syrup of need to regularly feeding, for drink clear water.
(6) results hybridization pod: in the time hybridizing pod maturation on test cross maternal plant, by combination and numbering results hybridization pod, JLCMS14B, as the community of top-cross male parent, gathers in the crops respectively 159,61 and 27 hybrid seeds on JLCMS101A, JLCMS9A and JLCMS31A; JLCMSR2 is as the community of top-cross male parent, on JLCMS101A, JLCMS9A and JLCMS31A, gather in the crops respectively 166, seed, 51 and 31, S39 is as the community of top-cross male parent, gathers in the crops respectively 146, seed, 63 and 22 on JLCMS101A, JLCMS9A and JLCMS31A;
(7) Second Year is pressed combined grow hybridization F spring
1seed, 15 seeds of each combination sowing, flowering stage is to 10 of each combination F
1individual plant is about to open bud sampling, with 75% alcohol fixation, tests indoor I
2-KI decoration method is observed pollen abortion rate, and average pollen abortion rate be the results are shown in to table 2.
3 parts of test cross male parent F of table 2
1
pollen abortion rate statistics (%)
(8) test cross result, three test cross female parents are hybridized F with JLCMS14B as can be seen from Table 2
1pollen abortion rate all exceed 98.3%, determine that JLCMS14B is maintainer; JLCMSR2 can highly recover JLCMS101A, also can recover male sterile line JLCMS9A, but can not recover the maternal JLCMS31A of test cross, its F
1pollen abortion rate is up to 83.7%, can determine that JLCMSR2 is a moderate strength restorer; The maternal hybridization F with S39 of three test crosses
1pollen abortion rate all exceed 60%, but all do not reach highly sterile standard, can determine S39 neither restorer neither maintainer.
test example 3
Checking embodiment 3 measures the recovery type of RN type sterile cytoplasm maintainer and restorer.
(1) select 2 parts of materials to carry out test cross, be respectively JLCMS42B and JLCMSR16.
(2) field planting and bloom before field management, field is by 2 male parents to be measured of community sowing and test cross maternal JLCMS82A, JLCMS9A and JLCMS31A.Community one sowing order: the first row JLCMS82A, JLCMS42B and JLCMS31A, parallel the second behavior JLCMS31A, JLCMS42B and JLCMS9A, cave is apart from 40cm; With a line, to second community of interval 100cm sowing, sowing material is test cross female parent and JLCMSR16, and seeding method is with first community; Before blooming, farming method is with Soybean production field.
(3) before blooming to all test cross fathers, maternal listing mark, simultaneously on test cross community, solarium for test cross is set up in assembling, and all test cross fathers, female parent are covered in solarium.With reference to Fig. 3.
(4) the maternal pollen abortion rate of microscopy, chooses and is about to open bud at the maternal initial bloom stage of test cross, and 3 flowers are got in every strain, fixing for subsequent use with 70% ethanol; At the indoor I of experiment
2-KI decoration method is observed pollen abortion rate, pulls out pollen abortion rate lower than 98% plant.
(5) throw in clover and cut Ye Feng: at the sunny weather of test cross father, maternal full-bloom stage, 2-3 clover megachilid thrown in to the maternal pollination of test cross by each solarium.Throw in after clover megachilid, in 50 meters, test region and periphery thereof, ban use of the virose agricultural chemicals of clover megachilid, until pollination finishes.Clover cuts after Ye Feng throws in does not need extra labor management.
(6) results hybridization pod: in the time hybridizing pod maturation on test cross maternal plant, by combination and numbering results hybridization pod, JLCMS42B, as the community of top-cross male parent, gathers in the crops respectively 168,69 and 26 hybrid seeds on JLCMS82A, JLCMS9A and JLCMS31A; JLCMSR16 is as the community of top-cross male parent, gathers in the crops respectively 93, seed, 48 and 20 on JLCMS82A, JLCMS9A and JLCMS31A.
(7) Second Year is pressed combined grow hybridization F spring
1seed, 15 seeds of each combination sowing, be about to open bud sampling to 10 of each combination individual plants flowering stage, with 75% alcohol fixation, tests indoor I
2-KI decoration method is observed pollen abortion rate, and average pollen abortion rate be the results are shown in to table 3.
2 parts of test cross male parent F of table 3
1
pollen abortion rate statistics (%)
(8) test cross result, three test cross female parents are hybridized F with JLCMS42B as can be seen from Table 3
1pollen abortion rate all exceed 97.3%, determine that JLCMS42B is maintainer; And JLCMSR16 can recover the maternal JLCMS82A of test cross and JLCMS9A, but can not recover JLCMS31A, its F
1pollen abortion rate, up to 93.3%, can determine that JLCMSR16 is a weak restorer.
Claims (3)
1. the male sterile test cross method of efficient soya cells matter, is characterized in that:
Utilize simultaneously one group typical, dissimilar sterile be test cross female parent, under isolation condition, adopt the many female collocation methods of a father, utilize insect pollination, it is maintainer or restorer and different recovery types that a test cross can be measured detected materials.
2. the male sterile test cross method of efficient soya cells matter, comprises the following steps:
1) select test cross female parent:
The first test cross female parent: such male sterile line can be kept by all maintainers, can be recovered by all restorers F simultaneously
1pollen abortion rate is between 0-10%;
The second test cross female parent: such male sterile line can be kept by all maintainers, can be exceeded 50% restorer recovery simultaneously, F
1pollen abortion rate is between 11-60%;
The 3rd test cross female parent: can be kept by all maintainers, only can be less than 10% restorer simultaneously and recover, F
1pollen abortion rate is greater than 61%;
2) solarium for test cross:
Test cross is to form enclosure space by reinforcing bar square frame skeleton and the gauze cover that covers on it with solarium, and test cross is provided with zipper opening with the side gauze of solarium;
3) field sowing mode:
Sowing season, in land for growing field crops, by community sowing, three kinds of test crosses of step 1) are maternal forms a test cross community with 1 male parent to be measured, each community sowing 1-2 cave male parent to be measured and three kinds, 4-5 cave test cross female parent; Cell size be vertical ridge to long 80-130cm, parallel ridge is to wide 80-120cm; Seeding method is bunch planting, and the first ridge planting order is the first test cross female parent, male parent to be measured and the 3rd test cross female parent, and the second ridge planting order is the second test cross female parent, male parent to be measured and the 3rd test cross female parent, and seeding quantity is 4, every cave, final singling 2 strains after emerging; According to suitably adjusting father, maternal sowing time in father, maternal flowering stage, consistent to guarantee father, maternal flowering stage; When the recovery capability of test cross restorer, can select 1-2 the high male sterile line sowing of coordinate force in community, to be hybrid with it a small amount of hybrid seed of preparation simultaneously;
4) set up solarium for test cross:
Within 3-5 days before test cross father, female parent are bloomed, give on each test cross community and set up a step 2) solarium for test cross, and remove wild insect in solarium;
5) male sterile line pollen abortion rate checks:
Button test cross, with before solarium, is given each test cross father, maternal listing mark, and button test cross is with behind solarium, by the maternal male sterile line pollen abortion of microexamination test cross rate, pulls out pollen abortion rate lower than 98% plant;
6) insect pollinator is thrown in:
At test cross father, maternal full-bloom stage, each test cross is thrown in 2-3 clover megachilid to the maternal pollination of test cross with solarium, for guaranteeing pollination quality, meets rainy weather and can repeat to throw in clover megachilid one time; Or with honey bees pollination, 100 left and right of bee colony size;
7) insect pollinator management:
Clover cuts after Ye Feng throws in does not need extra labor management; The honeybee syrup of need to regularly feeding, for drink clear water;
8) hybridization pod results:
On test cross maternal plant, hybridize after pod maturation, also record in detail seed source by combination and numbering results hybridization pod;
9) hybridization F
1plant pollen abortion rate checks: in next one plantation season, by combined grow hybridization F
1seed, by observing F
1plant pollen abortion rate, determines extensive guarantor's relation of male parent material to be measured, if restorer is determined the type of restorer.
3. the male sterile test cross method of efficient soya cells matter according to claim 2, is characterized in that:
The male sterile line of the first test cross female parent take JLCMS82A or JLCMS101A as representative; The male sterile line of the second test cross female parent take JLCMS9A as representative; The male sterile line of the 3rd test cross female parent take JLCMS89A or JLCMS31A as representative.
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| CN104004853A (en) * | 2014-06-18 | 2014-08-27 | 吉林省农业科学院 | Method for identifying soybean male sterile cytoplasm through SNP marks of chloroplast DNA |
| CN105532427A (en) * | 2015-12-02 | 2016-05-04 | 南京林业大学 | Net room apparatus and potting apparatus for preservation of woody fruit tree germplasm resources and preservation method |
| CN105875226A (en) * | 2016-04-17 | 2016-08-24 | 吉林省农业科学院 | Method for identifying fertility stability of cytoplasmic male sterile lines and hybrids of soybeans |
| CN110419444A (en) * | 2019-09-06 | 2019-11-08 | 中国农业科学院麻类研究所 | The method of entomophila hermaphrodite flower herbaceous plant insect pollination |
| WO2021027249A1 (en) * | 2019-08-15 | 2021-02-18 | 吉林省农业科学院 | Method for identifying outcrossing rate of self-pollination plant |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104004853A (en) * | 2014-06-18 | 2014-08-27 | 吉林省农业科学院 | Method for identifying soybean male sterile cytoplasm through SNP marks of chloroplast DNA |
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| CN105875226A (en) * | 2016-04-17 | 2016-08-24 | 吉林省农业科学院 | Method for identifying fertility stability of cytoplasmic male sterile lines and hybrids of soybeans |
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| WO2021027249A1 (en) * | 2019-08-15 | 2021-02-18 | 吉林省农业科学院 | Method for identifying outcrossing rate of self-pollination plant |
| CN110419444A (en) * | 2019-09-06 | 2019-11-08 | 中国农业科学院麻类研究所 | The method of entomophila hermaphrodite flower herbaceous plant insect pollination |
| CN115281073A (en) * | 2022-10-08 | 2022-11-04 | 潍坊市农业科学院(山东省农业科学院潍坊市分院) | Crossbreeding method for regulating flowering phase of clove |
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