CN102783416A - Breeding method for resistant super-male polyploidy asparagus based on flow cytometry - Google Patents
Breeding method for resistant super-male polyploidy asparagus based on flow cytometry Download PDFInfo
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- CN102783416A CN102783416A CN201210259546XA CN201210259546A CN102783416A CN 102783416 A CN102783416 A CN 102783416A CN 201210259546X A CN201210259546X A CN 201210259546XA CN 201210259546 A CN201210259546 A CN 201210259546A CN 102783416 A CN102783416 A CN 102783416A
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Abstract
The invention discloses a breeding method for resistant super-male polyploidy asparagus based on flow cytometry. The method comprises the following steps: (1) choosing excellent male plants in a field and carrying out indoor planting and microscopic examination; (2) mechanically separating microspores; (3) carrying out flow sorting; (4) carrying out suspension culture; (5) screening resistant mutants; (6) carrying out artificial doubling; (7) carrying out flow analysis; (8) subjecting the microspores obtained in step (7) respectively to field selective matching and cross combination and to in vitro rapid propagation; and (9) carrying out variety comparative tests on plants obtained after field selective matching and cross combination and plants obtained after in vitro rapid propagation, and applying plants obtained in the tests in production in the field. The method provided by the invention has the following beneficial effects: (1) a breeding period is shorter, breeding efficiency is higher, and operation is simpler; (2) an obtained variety has comprehensiveness; and (3) a breeding technology is novel.
Description
Technical field
The invention belongs to biological technical field, relate to the selection of the disease-resistant polyploidization supermale of a kind of asparagus strain, especially the suspension method for inducing and cultivating of the microspore resistance polyploid on streaming compartment analysis basis.
Background technology
The Isolated microspore cultivation is meant with methods such as machineries isolates the plant flowers powder that is in suitable breeding time; Carry out suspension culture on the liquid medium within; And then induce pollen embryo and haplobiont, finally handle the process that obtains the high ℃ double haploid offspring of isozygotying (DH colony) of genotype through artificial doubling.Compare with the method for passing through anther culture acquisition double haploid of routine, Isolated microspore is cultivated possesses clear superiority: genotypic influence has been reduced in (1), and induction frequency is high.Microspore is unicellular, and colony's quantity is big, and the radix that single is cultivated can reach hundreds thousand of, though induction frequency be ten thousand/, can guarantee that also the success of regeneration plant is induced.(2) microspore has been eliminated the interference of medicine wall tissue for exposed cell, and the offspring plant that is produced can not mix the liploid plant of inducing from medicine wall histocyte, population genetic background homogeneous.(3) microspore plasticity is strong, is easy to receive inducing of the culture environment factor, makes it to grow towards the direction of breeding man expectation.(4) operating technology is simple relatively, and separative efficiency is high.At present, this technology has been successfully applied to the modern biotechnology breeding fields such as cultivation, ploidy breeding, plant gene conversion, molecular marker assisted selection of resistant mutants screening, the inbred line of crops such as rape, barley, triticale.Become improve crop heterosis, fixedly crop hybrid high-quality, cultivate the effective tool of polyploidization pure line cultivar, resistant variety shortening breeding cycle.And be about to become the core technology of modern biotechnology breeding system.
Asparagus is the perennial perennial root herbaceous plant of Liliaceae, and dioecism is a product organ with tender stem, is the health-care vegetable that liked by the consumer.Under the natural conditions asparagus staminiferous plant to send out the stem number many, the PR female plant is high by about 25%, therefore complete male breeding has become the focus of domestic and international asparagus breeding research.The genotype of natural asparagus staminiferous plant is Mm, and the genotype of female plant is mm, obtain the F1 hybrid seed of offspring Quan Xiong; Can only rely on supermale strain MM and female plant mm to hybridize; Yet because the hereditary basis of asparagus is too complicated, obtaining supermale strain parent through selfing often needs the more than ten years even more of a specified duration, therefore; In conventional breeding in the past, will obtain very difficulty of a good complete male crossbreed, this also is the expensive main cause of asparagus seeds.In recent years; Continuous maturation along with modern tissue culture technique; The China scholar has done a large amount of and fruitful research to the tissue culture technique of asparagus, has successfully induced the asparagus regeneration plant, and has carried out the ploidy breeding research on anther culture, the detoxification culture technique basis.Make the rearing new variety level of China asparagus reach international most advanced level.Yet because the cultivation defective that anther culture can not be eliminated, the efficient that obtains haplobiont and excellent strain is not high.
Some early stage UC800 that introduce, UC72, Mary Washington etc. mostly are external kind of eliminating already, yield poorly, a little less than the disease resistance, poor quality, it is long to lead and form the required time of economic flow rate; Grow seedlings then generally speaking, transplanted in the 2nd year, the 3rd year just begins to gather; Grow seedlings then with Hybrid; Transplant then, can gather in the 2nd year and to compare, evening 1 year.Asparagus is perennial perennial root vegetables, and once cultivation can be gathered 15 years continuously, so the quality of its kind will influence the yield and quality of asparagus whole life, and determined the success or failure of its cultivation.Begin from the eighties in 20th century; Each R&D institution of China has carried out the research of asparagus breeding in succession; Along with development of biology such as tissue culture; China asparagus breeding research person utilizes modern biotechnology to combine with conventional breeding, obtained bigger scientific achievement and progress, has cultivated some high yields, high-quality, disease-resistant asparagus new varieties.At present, the single-strain clone crossbreeding is the main method of China's asparagus breeding: promptly through introducing the asparagus variety source, according to breeding goal; Filter out have various merits female, staminiferous plant as hybrid strain, assembly hybrid combination compares evaluation to F1 again; Filter out good hybrid combination, and combine group culturation rapid propagating technology, expand numerous two parents' single-strain clone; Utilize female, male parent clone to carry out the scale production of hybrid seeds, can breed the new varieties (being) that meet breeding goal.In addition; The researcher on ground such as Shandong, Jiangxi also utilizes colchicine to handle the supermale strain and obtains the strain of tetraploid supermale; The method of cultivating 3 times of full staminiferous plants of body with the method for dliploid female plant hybridization has again been carried out the research of asparagus polyploid breed breeding; And cultivated J2-2, the purple P-7 in the Weihe River, 3 times of body new varieties of the red grade in well ridge.But above-mentioned kind do not have possess salt tolerant, drought-enduring, characteristic such as impoverishment tolerant is disease-resistant.
Summary of the invention
The objective of the invention is to overcome the shortcoming of above-mentioned prior art; Providing a kind of is explant with the free microspore of manual work; Anti-stem is withered to cultivate, the strain of salt tolerant polyploid supermale is a purpose, and the biological new technology of breeding of various modern such as integrated use microspores culture, disease-resistant salt-tolerant mutant screening, artificial doubling and streaming ploidy analysis is cultivated the method for asparagus kind.
The objective of the invention is to solve through following technical scheme:
A kind of asparagus resistance supermale polyploidization breeding method based on the cell streaming technology, according to following steps:
(1) selects the good staminiferous plant in land for growing field crops, carry out indoorly planting, microscopy;
(2) mechanical separation microspore;
(3) microspore that mechanical separation is gone out carries out airflow classification;
(4) microspore that sub-elects of convection type carries out suspension culture;
(5) step (4) is cultivated the pollen embryo of inducing and carry out the resistant mutants screening;
(6) the monoploid resistant plant that step (5) is obtained carries out artificial doubling to be handled;
(7) regeneration plant that doubles that step (6) is obtained carries out flow cytometer showed;
(8) the tetraploid supermale system that step (7) is obtained carries out the field apolegamy hybrid combination and the breeding fast of exsomatizing respectively;
(9) breeding gained plant carries out variety comparative test with exsomatizing fast the strain of hybrid combination gained triploid resistance supermale to be matched in the field; And the experiment sieving elite plant strain is used for variety certification and land for growing field crops promotes.
Said step (1) is to select land for growing field crops high yield, consistent, the good staminiferous plant of no damage by disease and insect of tender tips of bamboo shoot size, carries out indoor plant, collect not open Xiao Hua, microscopy, selects form consistent, is in monokaryotic stage Xiao Hua.
Said step (2) be earlier with microspore at 10 ℃ of preliminary treatment 7d+ caseinhydrolysates, mechanical then homogenate 7s, at 9600rpm twice filters, the 200g washing, centrifugal collection microspore.
Said step (3) is with the analysis of recovery microspore aseptic condition downflow system, and according to forward angle light scatter light, the E type pollen piezoelectricity that volume is bigger sorts out.
Said step (4) is that the microspore that convection type sub-elects carries out suspension culture, and 25 ℃ of NBP99+10%FICOIL secretly cultivate 21-35d.
Said step (5) is to adopt MES differential medium+variable concentrations resistant mutants toxin screening pressure, 18 ℃, 12000lx, progressively induction of resistance haplobiont.
Said step (6) is to adopt concentration of volume percent 0.08% colchicine to dip in root artificial doubling haplobiont seedling stage.
Said step (7) is to use streaming dna content analytical technology, according to cell nucleus relative intensity of fluorescence difference between the Different Ploidy plant, the tetraploid plant mirror that doubles in the colony of back is elected.
Said step (8) is that gained tetraploid individual plant is carried out agronomic performance evaluation, with disease-resistant good high yield individual plant and the good female plant of dliploid combine measured coordinate force in twos.
Said step (9) is identified economical character and varietal yield test with crossbreed sowing and land for growing field crops, and fine individual plant is used for the new varieties evaluation and promotes.
The invention has the beneficial effects as follows: the explant that (1) is different.With the microspore is that explant has the advantage that many flower pesticide do not possess, and efficient is higher, operation is simpler, and international and domestic still the expansion similarly studied and used.(2) technological synthesis property is strong.Combine various modern biotechnology breeding new technology, breeding cycle is shorter, and efficient is higher.(3) kind is comprehensive strong.The kind of seed selection will integrate multiple good characteristic, rather than the detoxifying fast breeding on the simple meaning.(4) breeding technique is new.
Description of drawings
Fig. 1 is the haploid method flow diagram of microspores culture efficient induction asparagus of the present invention.
Embodiment
Below in conjunction with accompanying drawing the present invention is done and to describe in further detail:
Referring to Fig. 1, a kind of asparagus resistance supermale polyploidization breeding method based on the cell streaming technology, according to following steps:
(1) selects the good staminiferous plant in land for growing field crops (consistent, the no damage by disease and insect of high yield, tender tips of bamboo shoot size), carry out indoorly planting, microscopy (collect open Xiao Hua, microscopy, select the form unanimity, be in monokaryotic stage Xiao Hua);
(2) mechanical separation microspore (10 ℃ of preliminary treatment 7d+ caseinhydrolysates) (mechanical homogenate 7s 9600rpm twice, filtration, 200g washing, centrifugal collection microspore);
(3) microspore that mechanical separation is gone out carries out airflow classification; Said step (3) is with the analysis of recovery microspore aseptic condition downflow system, and according to forward angle light scatter light, the E type pollen piezoelectricity that volume is bigger sorts out.
(4) microspore that sub-elects of convection type carries out suspension culture (25 ℃ of NBP99+10%FICOIL secretly cultivate 21-35d);
(5) step (4) is cultivated the pollen embryo of inducing and carry out the resistant mutants screening; Said step (5) is to adopt MES differential medium+variable concentrations resistant mutants toxin screening pressure, 18 ℃, 12000lx, progressively induction of resistance haplobiont.
(6) the monoploid resistant plant that step (5) is obtained carries out artificial doubling and handles (colchicine artificial doubling); Said step (6) is to adopt concentration of volume percent 0.08% colchicine to dip in root artificial doubling haplobiont seedling stage.
(7) regeneration plant that doubles that step (6) is obtained carries out flow cytometer showed; Said step (7) is to use streaming dna content analytical technology, according to cell nucleus relative intensity of fluorescence difference between the Different Ploidy plant, the tetraploid plant mirror that doubles in the colony of back is elected.
(8) the tetraploid supermale system that step (7) is obtained carries out the field apolegamy hybrid combination and the breeding fast of exsomatizing respectively; Said step (8) is that gained tetraploid individual plant is carried out agronomic performance evaluation, with disease-resistant good high yield individual plant and the good female plant of dliploid combine measured coordinate force in twos.
(9) breeding gained plant carries out variety comparative test with exsomatizing fast the strain of hybrid combination gained triploid resistance supermale to be matched in the field; And the experiment sieving elite plant strain is used for variety certification and land for growing field crops promotes.Said step (9) is identified economical character and varietal yield test with crossbreed sowing and land for growing field crops, and fine individual plant is used for the new varieties evaluation and promotes.
The airflow classification of A pollen grain: the pollen of any plant all has dimorphism; It is often bigger that some are in the inner pollen grain individuality of flower pesticide; And that periphery is pressed close to the pollen grain of tapetum is less; These pollen grains that vary in size are being induced on the efficient of pollen plant widely differently, and little pollen grain is easy concerning some plant, and the plant that has is just in time opposite.Utilization airflow classification technology according to the size of forward angle light scatter light area, is cultivated the efficient of inducing that will improve pollen plant greatly with different pollen grains separation.
The mechanical type of B pollen grain separates: early stage in the research of other plant, extrusion commonly used is separated pollen grain and is cultivated, however excessive physical pressure usually the precocity pollen grain break or injured; It is very poor to cultivate reaction; Separate microspore with mechanical type and the method that is aided with density gradient centrifugation, not only separative efficiency improves greatly, also can be reduced to minimum degree to the damage of microspore; Simultaneously can fall living cells and carry out preliminary separating, more help to improve and induce efficient with dead cell.
C microspores culture and resistant mutants screening: microspore does not form at ripe prepollen outer wall; There is not the formation of cell wall yet; Whole microspore is only encapsulating the very thin cell membrane of one deck, owing to lacked the obstruct of cell wall, and extraneous trophic factors, hormone and coerce the factor and be easy to affact pollen grain inside; Induce pollen grain to change, make it will seek development according to the breeding people.As in medium, progressively adding the salt of different gradient concentrations, the stem wilt verticillium toxin is constantly tamed cell, and final formation has disease-resistant new strain with salt tolerant and is.
The D ploidy breeding: a lot of liploid plants all can cause the organ gigantism after doubling, will be like the output of spinach, sugarbeet tetraploid kind far above dliploid, and triploid seedless watermelon yield also usually is higher than dliploid.Natural asparagus staminiferous plant genotype is Mm, carries out can obtaining M or m monoploid after pollen is cultivated, and it is individual to obtain dliploid MM (supermale strain) and mm after once doubling, and they carry out complementary hybridization with the MM of other kinds and mm plant can obtain the F1 hybrid of hero entirely., MM and mm plant can obtain tetraploid MMMM plant and mmmm plant after doubling once more; When MMMM and the hybridization of mm plant, just can obtain the sterile superpower staminiferous plant MMm of triploid; Because the nutrition saving that the increase of ploidy and reproductive growth disappearance cause will make the complete male asparagus of triploid possess higher yield and quality; We are also unknown whether can further to promote output as for the hybrid of Hyperploidy more, attempt but still be worth exploring.
The E streaming is identified: flow cytometry is a cell particle high speed analysis, the sorting technology that last century, the eighties grew up, and has been widely used in medical domain.Its principle is that the cell of Different Ploidy possesses the nuclear DNA that contains different sizes; After these DNA are by DNA specific bond property fluorescent dyeing; After the exciting of exciting light, can produce autofluorescence; The size of how much examining ploidy of nuclear dna content is also being represented in the power of autofluorescence and how much positive correlation of fluorescent dye.When these cell particles that are colored through a test point time, the cells of different sizes will be identified and sorting.Its smart ℃ can reach the chromosomal difference of plate half bar; After the asparagus regeneration plant is doubled; How the individual ploidy of the numerous regeneration of fast detecting just becomes very urgent, and the flow cytometer showed technology provides method very easily for us, and it can detect up to a hundred plant in a working day; And if adopt conventional cell compressing tablet chromosome numeration method to identify, the evaluation that accomplish a material often needs one to several weeks.
The above only is preferred embodiment of the present invention, is not the present invention is done any pro forma restriction; Though the present invention discloses as above with preferred embodiment; Yet be not in order to limiting the present invention, anyly be familiar with the professional and technical personnel, in not breaking away from technical scheme scope of the present invention; When the method for above-mentioned announcement capable of using and technology contents are made a little change or be modified to the equivalent embodiment of equivalent variations; In every case be the content that does not break away from technical scheme of the present invention, to any simple modification, equivalent variations and modification that above embodiment did, still belong in the scope of technical scheme of the present invention according to technical spirit of the present invention.
Claims (10)
1. the asparagus resistance supermale polyploidization breeding method based on the cell streaming technology is characterized in that, according to following steps:
(1) selects the good staminiferous plant in land for growing field crops, carry out indoorly planting, microscopy;
(2) mechanical separation microspore;
(3) microspore that mechanical separation is gone out carries out airflow classification;
(4) microspore that sub-elects of convection type carries out suspension culture;
(5) step (4) is cultivated the pollen embryo of inducing and carry out the resistant mutants screening;
(6) the monoploid resistant plant that step (5) is obtained carries out artificial doubling to be handled;
(7) regeneration plant that doubles that step (6) is obtained carries out flow cytometer showed;
(8) the tetraploid supermale system that step (7) is obtained carries out the field apolegamy hybrid combination and the breeding fast of exsomatizing respectively;
(9) breeding gained plant carries out variety comparative test with exsomatizing fast the strain of hybrid combination gained triploid resistance supermale to be matched in the field; And the experiment sieving elite plant strain is used for variety certification and land for growing field crops promotes.
2. asparagus resistance supermale polyploidization breeding method as claimed in claim 1; It is characterized in that: said step (1) is to select land for growing field crops high yield, consistent, the good staminiferous plant of no damage by disease and insect of tender tips of bamboo shoot size; Carry out indoor plant, collect not open Xiao Hua, microscopy; The selection form is consistent, is in monokaryotic stage Xiao Hua.
3. asparagus resistance supermale polyploidization breeding method as claimed in claim 1; It is characterized in that: said step (2) be earlier with microspore at 10 ℃ of preliminary treatment 7d+ caseinhydrolysates; Mechanical then homogenate 7s, at 9600rpm twice, filter, the 200g washing centrifugal collection microspore.
4. asparagus resistance supermale polyploidization breeding method as claimed in claim 1 is characterized in that: said step (3) is with the analysis of recovery microspore aseptic condition downflow system, and according to forward angle light scatter light, the E type pollen piezoelectricity that volume is bigger sorts out.
5. asparagus resistance supermale polyploidization breeding method as claimed in claim 1 is characterized in that: said step (4) is that the microspore that convection type sub-elects carries out suspension culture, and 25 ℃ of NBP99+10%FICOIL secretly cultivate 21-35d.
6. asparagus resistance supermale polyploidization breeding method as claimed in claim 1 is characterized in that: said step (5) is to adopt MES differential medium+variable concentrations resistant mutants toxin screening pressure, 18 ℃, 12000lx, progressively induction of resistance haplobiont.
7. asparagus resistance supermale polyploidization breeding method as claimed in claim 1 is characterized in that: said step (6) is to adopt concentration of volume percent 0.08% colchicine to dip in root artificial doubling haplobiont seedling stage.
8. asparagus resistance supermale polyploidization breeding method as claimed in claim 1; It is characterized in that: said step (7) is to use streaming dna content analytical technology; According to cell nucleus relative intensity of fluorescence difference between the Different Ploidy plant, the tetraploid plant mirror that doubles in the colony of back is elected.
9. asparagus resistance supermale polyploidization breeding method as claimed in claim 1; It is characterized in that: said step (8) is that gained tetraploid individual plant is carried out agronomic performance evaluation, with disease-resistant good high yield individual plant and the good female plant of dliploid combine measured coordinate force in twos.
10. asparagus resistance supermale polyploidization breeding method as claimed in claim 1 is characterized in that: said step (9) is identified economical character and varietal yield test with crossbreed sowing and land for growing field crops, and fine individual plant is used for the new varieties evaluation and promotes.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103529202A (en) * | 2013-09-13 | 2014-01-22 | 中国科学院合肥物质科学研究院 | Method for reducing spontaneous mutation background of wild type human-rat hybridoma AL cells |
| CN103749293A (en) * | 2013-12-20 | 2014-04-30 | 江西省农业科学院 | Method for obtaining regenerated plantlet through anther culture of asparagus officinalis and asparagus dauricus interspecific crossing F1 generation |
| CN105766648A (en) * | 2016-04-06 | 2016-07-20 | 西北农林科技大学 | Method for screening anti-black rot mutant plants with cabbage free microspores |
| CN110542641A (en) * | 2019-09-26 | 2019-12-06 | 华南农业大学 | Method for sorting 2n pollen from pollen |
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2012
- 2012-07-25 CN CN201210259546XA patent/CN102783416A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103529202A (en) * | 2013-09-13 | 2014-01-22 | 中国科学院合肥物质科学研究院 | Method for reducing spontaneous mutation background of wild type human-rat hybridoma AL cells |
| CN103529202B (en) * | 2013-09-13 | 2015-04-15 | 中国科学院合肥物质科学研究院 | Method for reducing spontaneous mutation background of wild type human-rat hybridoma AL cells |
| CN103749293A (en) * | 2013-12-20 | 2014-04-30 | 江西省农业科学院 | Method for obtaining regenerated plantlet through anther culture of asparagus officinalis and asparagus dauricus interspecific crossing F1 generation |
| CN103749293B (en) * | 2013-12-20 | 2015-09-30 | 江西省农业科学院 | Asparagus and Xingan's asparagus interspecific cross F 1for the method for anther culture regeneration plant |
| CN105766648A (en) * | 2016-04-06 | 2016-07-20 | 西北农林科技大学 | Method for screening anti-black rot mutant plants with cabbage free microspores |
| CN110542641A (en) * | 2019-09-26 | 2019-12-06 | 华南农业大学 | Method for sorting 2n pollen from pollen |
| CN110542641B (en) * | 2019-09-26 | 2020-11-06 | 华南农业大学 | Method for sorting 2n pollen from pollen |
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Application publication date: 20121121 |