CN102144560B - Method and application method for obtaining novel germ plasm of brassica A genome vegetable - Google Patents
Method and application method for obtaining novel germ plasm of brassica A genome vegetable Download PDFInfo
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Abstract
The invention discloses a method and application method for obtaining a novel germ plasm of brassica A genome vegetable. In the method, free microspore culture is carried out on crossbreeds among vegetables of Chinese cabbage subspecies*pakchoi cabbage subspecies or variety in pakchoi cabbage subspecies*variety of the brassica A genome to obtain a regeneration plant and further to create the novel germ plasm which is used for breeding. The method mainly comprises the following steps of: hybridizing the vegetables of Chinese cabbage subspecies*pakchoi cabbage subspecies or variety in pakchoi cabbage subspecies*variety; inducing free microspore culture embryoids; germinating the embryoids to obtain the regeneration plant; carrying out ploidy identification on the regeneration plant obtainedby microspore culture; reserving seeds of double haploids and polyploids; planting an offspring of the regeneration plant to carry out morphological identification and nutritive index identification;and preparing, hybridizing and combining. The method has the advantages of short cycle and high efficiency, provides a material for efficiently breeding Chinese cabbage vegetables and has broad application prospect.
Description
Technical field
The invention belongs to biotechnology and haploid breeding technical field, specifically, relate to and a kind ofly obtain the method that rape belongs to A genome vegetables new germ plasm and is applied to breeding fast by the Isolated microspore cultural method.
Background technology
Rape belongs to A genome (2n=2x=20) vegetables, be divided into a variety of Chinese cabbage subspecies and Chinese cabbage subspecies, the a variety of Chinese cabbage subspecies comprise that milk Chinese cabbage, Wuta-tsai, cabbage heart, little Chinese cabbage, rape, short pin Huang, flower bud are with dish etc., the characteristics of these subspecies are that blade is opened a business, short and small, the tangible petiole of tool of plant type, the smooth nothing hair of most blades, do not have the obvious leaf wing, do not form obvious leaf-head.The Chinese cabbage subspecies are divided into the leaf mutation half hitch ball mutation of loosing, mutation fickle in love, balling mutation, and characteristics are that plant type is bigger, and tangible petiole and Ye Yi are arranged, and how rough the blade face is, except diffusing leaf mutation, all can form leaf-head.Between the subspecies and in the subspecies between the mutation vegetables proterties widely different because chromosome bar number is the same, between the subspecies and in the subspecies between the mutation hybridization do not have obstacle, easily obtain seed.The advantage of different subspecies, mutation vegetables can be integrated by sexual hybridization, filial generation genotype be highly mix and, and the pure and mild parent of the height that needs in the advantage crossbreeding with the conventional method selfing, separate and select selfing continuously several from generation to generation again, takes a lot of work, time-consuming.Raising along with people's living standard, conventional method is interior with mutation, intervarietal cross, be difficult to satisfy the requirement of vegetables of high yield, high-quality, the shape novelty of market demand, to between the subspecies and in the subspecies between the mutation hybrid carry out Isolated microspore and cultivate and then can obtain the double haploid pure lines fast, and it is widely different between the pure lines, bigger range of choice is provided, has addressed this problem, satisfied the needs of breeding work.
Up to now, belong to the report that the microspores culture success is all arranged on the crop nearly all rape, 1989, Japan scholar Sato etc. is carrying out having obtained regeneration plant when the Chinese cabbage Isolated microspore is cultivated, and 1992, reported first a variety of Chinese cabbage Isolated microspores such as Cao Mingqing were cultivated and succeedd, 1996, Li Guangtao has obtained the purple tsai-tai regeneration plant, and in addition, Wuta-tsai etc. also have cultivates successful report.Based on rape belong between the A genome crop subspecies and in the subspecies mutation vegetables intermolecular hybrid do not have obstacle and in the progress that obtains aspect the microspores culture, the present invention belongs to ' Chinese cabbage subspecies * a variety of Chinese cabbage subspecies ', ' mutation * mutation in a variety of Chinese cabbage subspecies ' crossbreed as the donor plant of microspores culture with rape, the microspores culture system is carried out a large amount of tests, obtain a lot of regeneration plants, prepared hybrid combination.
Summary of the invention
The purpose of this invention is to provide a kind of method and application method that rape belongs to A genome vegetables new germ plasm that obtain, be that traditional sexual hybridization technology and cell culture technology are combined, carry out between the subspecies and subspecies in vegetables hybridization between the mutation, by the Isolated microspore cultural method to F
1Or the F that meets breeding objective through screening
2Cultivate, thereby obtain regeneration plant, and be applied to breeding.
The method that acquisition rape provided by the invention belongs to A genome vegetables new germ plasm is as follows:
A, rape is belonged to ' Chinese cabbage subspecies * a variety of Chinese cabbage subspecies ' or ' mutation * mutation in a variety of Chinese cabbage subspecies ' vegetables hybridize and obtain F
1Seed, plantation F
1And selfing obtains F
2
B, in the greenhouse, cultivate F
1And F
2, F
1And F
2Seed through 2～4 ℃ of low temperature treatment 20～25 days, flowering stage to F
1And F
2The plant that meets breeding objective that filters out in generation carries out Isolated microspore and cultivates, need 33 ℃ of dark cultivations 1 day behind the inoculation microspore, go to 25 ℃ of incubators again and leave standstill dark cultivation 10～15 days, behind the visible embryoid of naked eyes, it is to continue dark the cultivation on 40～60 rev/mins of shaking tables that culture dish is transferred to revolution, and cultivation temperature is 25 ℃;
C, the cotyledon period embryoid is transferred to the MS medium to obtain regeneration plant;
D, regeneration plant is inoculated into the enterprising pedestrian worker's root induction of MS medium, treat that regeneration plant is taken root after, be transplanted to the greenhouse, greenhouse temperature should be 20～25 ℃ daytime, nocturnal temperature should be in 5～10 ℃;
E, after 50～70 days, regeneration plant begins to bloom, and identifies the regeneration plant ploidy, haplobiont is handled with colchicin, and it is doubled, double haploid, high power body plant keep;
F, to double haploid, high power body plant pollination self, obtain seed, measure index of affinity simultaneously;
G, plantation double haploid, high power body selfed seed according to breeding objective, are identified appearance characters such as plant plant height, leaf length, leaf look, calcium, magnesium, all nutritive indexs of vitamin C, disease-resistant, resistance.
In the B step, to F
1And F
2Plant carries out Isolated microspore and cultivates, choose petal length and flower pesticide length ratio and be 0.5～0.75 open bud, after the sterilization of superclean bench inner surface, with distillation water flushing 3 times, pour the B5 medium that sucrose concentration is 130g/L into, squeeze the design of scattered small flowers and plants flower bud, make microspore dissociate out, contain the suspension of microspore with the nylon net filter in 400 order apertures, collect filtrate in the 10mL centrifuge tube, the centrifugal 3min of 1000rpm; Abandoning supernatant repeats 2 times; With NLN medium dilution microspore, inoculating cell density 1 * 10
5～2 * 10
5Individual mL
-1, to pack in the aseptic glass culture dish of 60mm * 15mm with every ware 5mL microspore suspension branch, every ware adds the agarose 0.5gL behind the 100 μ L high-temperature heat sterilizations
-1, active carbon 10gL
-1Mixed liquor; The NLN medium does not have the NLN medium of any hormone for containing 130g/L sucrose, or adds the 6-BA of 0.05mg/L～0.2mg/L at the NLN medium, or adds 6-BA and the NAA of 0.05mg/L～0.2mg/L at the NLN medium.
In the C step, the MS medium of regeneration induction plant is for containing sucrose 30g/L, agar 6.5g/L～7.5g/L, and the pH value is 5.8～6.0, does not have the MS medium of any hormone; In the D step, root media is the MS medium, contains sucrose 30g/L, agar 5.0g/L, and NAA0.1～0.2mg/L, the pH value is 5.8～6.0.
Rape provided by the invention belongs to the application method of A genome vegetables new germ plasm: the double haploid strain is directly used as kind, or as parent and the combination of male sterile line preparing hybrid, perhaps selected two self incompatible line preparing hybrid combinations.
The invention has the beneficial effects as follows: 1, compare with the conventional method intravarietal crossing, adopt rape to belong to ' Chinese cabbage subspecies * a variety of Chinese cabbage subspecies ' or ' mutation * mutation in a variety of Chinese cabbage subspecies ' hybridization, can obtain the vegetables newtypes that have utilization to be worth more.2, the method with conventional continuous multi-generation selfing obtains pure lines relatively, and it is short that the Isolated microspore cultured method is cultivated the pure lines cycle, the efficient height, and a generation just can obtain the double haploid pure lines.3, the pure lines differences of Isolated microspore method acquisition is bigger, has improved the probability that obtains to meet the breeding objective pure lines.4, the pure lines of the present invention's acquisition have broad application prospects.
Description of drawings
Fig. 1 is parent's Wuta-tsai form photo schematic diagram.
Fig. 2 is parent Chinese cabbage form photo schematic diagram.
Fig. 3 is the crossbreed F of ' Chinese cabbage and Wuta-tsai '
1(microspores culture donor material) form photo schematic diagram.
Fig. 4 is the crossbreed F of ' Chinese cabbage and Wuta-tsai '
1One of the regeneration plant that microspores culture obtains form photo schematic diagram.
Fig. 5 is the crossbreed F of ' Chinese cabbage and Wuta-tsai '
1Two form photo schematic diagram of the regeneration plant that microspores culture obtains.
Fig. 6 is the crossbreed F of ' Chinese cabbage and Wuta-tsai '
1Three form photo schematic diagrames of the regeneration plant that microspores culture obtains.
Embodiment
The method that acquisition provided by the invention rape belongs to A genome vegetables new germ plasm and is applied to breeding comprises: ' Chinese cabbage subspecies * a variety of Chinese cabbage subspecies ', ' mutation * mutation in a variety of Chinese cabbage subspecies ' crossover process, the Isolated microspore incubation, the regeneration plant ploidy that microspores culture obtains is identified, to double haploid, the high power body process of reserving seed for planting, the offspring of plantation regeneration plant carries out form evaluation, nutritive index qualification process, preparing hybrid anabolic process.
The concrete grammar step is as follows:
A, rape belong to ' Chinese cabbage subspecies * a variety of Chinese cabbage subspecies ' or ' mutation * mutation in a variety of Chinese cabbage subspecies ' crossover process: according to breeding objective, determine the parent, hybridization in flowering stage obtains F
1Seed, plantation F
1, selfing obtains F
2The parent is chosen to be: Chinese cabbage (seeing Fig. 2 photo), Wuta-tsai (seeing Fig. 1 photo), flower bud dish, short pin Huang.
The Isolated microspore culture materials is respectively ' Chinese cabbage * Wuta-tsai ' (seeing Fig. 3 photo), ' Wuta-tsai * flower bud dish ' and ' Wuta-tsai * short pin Huang ', F
1Generation and F
2For plant.
B, Isolated microspore incubation:
At the beginning of 2009 9 months, every other month in batches to F
1And F
2Presprouting of seeds was carried out 2～4 ℃ of low temperature treatment 20～25 days after sprouting, the seed of will sprouting then is sowed at the cave dish, plants during 5～6 leaves and cultivates in the greenhouse in flowerpot.Carry out Isolated microspore in flowering stage and cultivate, it is 0.5～0.75 open bud that the microspore separating method is chosen petal length and flower pesticide length ratio, with 30 be one group, flowing water washes 30min, behind 70% ethanolic solution surface sterilization 30s, uses 0.1%HgCl
2Solution disinfection 8min uses distilled water flushing 3 times, each 5min.Bud after the sterilization is put into the small beaker of the bacterium of going out, adds the B that a little sugar content is 130g/L
5Medium rolls bud with aseptic pestle, extrudes microspore.Contain the suspension of microspore with the nylon net filter in 40 μ m apertures, remove out and organize piece greatly, collect filtrate in the 10mL centrifuge tube, centrifugal 3min under the 1000rpm.Abandoning supernatant, sediment adds 5mLB
5The washing medium shakes up centrifugal 3min under the 1000rpm.Abandoning supernatant repeats 2 times, and the gained sediment is pure microspore.Microspores culture: the microspore behind the purifying with the dilution of NLN medium, is kept cell density 1 * 10
5～2 * 10
5Individual mL
-1, packing into every ware 5mL microspore suspension branch is in the aseptic glass culture dish of 60mm * 15mm, every ware adds agarose 0.5g/L, the active carbon 10g/L mixed liquor behind the 100 μ L high-temperature heat sterilizations; Seal with Parafilm, in 33 ℃ of insulating boxs, secretly cultivated 1 day earlier,, go to again and leave standstill dark cultivation under 25 ℃.After 10～15 days, the visible embryoid of naked eyes occurs, and transfers to and continues dark the cultivation on 40～60 rev/mins of shaking tables, and cultivation temperature is 25 ℃.The NLN medium is for containing 130g/L sucrose, the NLN medium that does not have any hormone, or add the 6-BA (6-benzyl aminoadenine) of 0.05mg/L～0.2mg/L at the NLN medium, or add 6-BA and the NAA (α-Nai Yisuan) of 0.05mg/L～0.2mg/L at the NLN medium.
C, plant regeneration process
The cotyledon period embryoid is inoculated on the MS medium.In 25 ℃, the 16h/8h photoperiod cultivates down.The MS medium is for containing sucrose 30g/L, agar 6.5g/L～7.5g/L, and the pH value is 5.8～6.0, does not have the MS medium of any hormone, obtains regeneration plant.
The taking root and transplant of D, regeneration plant
The regeneration plant that the microspore embryoid forms, the root system of taking root naturally a little less than, be difficult for surviving, need carry out the artificial induction and take root, it is inoculated on the MS medium, the MS medium contains sucrose 30g/L, agar 5.0g/L, NAA0.1～0.2mg/L, the pH value is 5.8～6.0.
Root system development preferably plantlet of transplant to the greenhouse.At this moment, temperature should be 20～25 ℃ daytime in the greenhouse, and evening temperature should be 5～10 ℃;
E, ploidy qualification process
After 50～70 days, regeneration plant begins to bloom, and identifies the regeneration plant ploidy, and haplobiont is handled with colchicin, and it is doubled, and double haploid, high power body plant keep; Adopt the ploidy of the method judgement regeneration plant that the plant external form is observed and pollen fertility is identified.When pollen vigor and pollen grain size are identified, get the bud that is about to bloom, strip flower pesticide and place on the clean slide, add 1～2 TTC (2,3,5-triphenyltetrazolium chloride) dye liquor, extrude pollen with tweezers, remove impurity, stir evenly the back covered, place 35 ℃ of insulating boxs, microscopy behind 10～15min, get 3 visuals field at random for every, add up in each visual field the pollen grain sum and incarnadine the pollen grain number, calculate the rate of dyeing of pollen.The pollen viability that reddens through the dyeing of TTC dye liquor is strong, is the fertile flower powder; Faint yellow and colourless vigor is more weak or do not have vigor, is pollen sterile.
F, to double haploid, high power body plant pollination self, obtain seed, measure index of affinity simultaneously;
G, plantation double haploid, high power body selfed seed according to breeding objective, are identified appearance characters such as plant plant height, leaf length, leaf look, nutritive indexs such as calcium, magnesium, vitamin C, disease-resistant, resistance;
Application method: the needs according to market are directly used the double haploid strain as kind, or as parent and the combination of male sterile line preparing hybrid, perhaps select two self incompatible line preparing hybrid combinations.
Result of the test:
1, homogenic type hybrid combination microspores culture embryonal induction result
Genotype is to influence the key factor that the microspore embryo takes place, and when medium all is NLN, all obtained embryoid for the examination material, but different parent is hybridized the F of gained
1For the inductivity of microspore embryo widely different (table 1), F
2In generation, do not done statistical analysis owing to embryoid induction rate difference between each individual plant.
The different donor materials of table 1 are to the influence of microspore embryoid induction rate
Annotate: data are 3 repetition mean values in the table, and Duncan multiple range test, different lowercases are that difference reaches extremely significantly (α=0.05)
2, plant growth regulator is to the influence of embryoid generation
Add 6-BA or 6-BA and NAA proportioning and be conducive to the embryoid generation, the suitableeest concentration is ' 0.1mg/L6-BA+0.05mg/L NAA ' (table 2).
The influence that table 2 plant growth regulator takes place ' Wuta-tsai * flower bud dish ' embryoid
Annotate: data are 3 repetition mean value Duncan multiple range tests in the table, and different lowercases are that difference reaches extremely significantly (α=0.05)
3, embryoid is sprouted, the acquisition of regeneration plant
In order to obtain a large amount of double haploids, carried out test of many times, obtain 664 of embryoids altogether, forward in the MS medium, embryoid continues to grow, and forms regeneration plant 375 strains.
4, the influence of the rooting of vitro seedling of plant growth regulator NAA
When ambient temperature is suitable, the greenhouse is taken root, is transplanted to test-tube plantlet.On the medium of MS, add NAA0.1mg/L～0.15mg/L, cultivating 12d just has root to occur, and root system is strong, and quantity is many.The regeneration plant of unrooted is along with the increase meeting of subculture number is taken root naturally, but such root is generally very thin and delicate, transplants to be difficult for surviving, and needs to add hormone induction and takes root.By the regeneration plant rooting rate be comparison shows that, with the culture of rootage effect best (table 3) of NAA 0.1mg/L.The microspore plant that how much influences of agar content is transplanted, and agar content is few, and medium is then softer, washes the root ratio during transplanting and is easier to, and agar content is many, and medium is hard, washes Gen Shiyi fibrous root is broken.Through test, determine that " MS+NAA 0.1mg/L+30g/L sucrose+5.0g/L agar " is the appropriate media that microspore plant is taken root.
The influence of the rooting of vitro seedling of table 3NAA
5, the regeneration plant ploidy is identified
Adopt the ploidy of the method judgement regeneration plant that the plant external form is observed and pollen fertility is identified.A little less than the haplobiont growing way, bud is thin and weak, and flower pesticide is shrivelled, and pollen is few.The double haploid stalwartness, flower bud development is good, and pollen is more.High power body plant bud is very large, is prone to split the flower bud phenomenon, and pollen is big and out-of-shape than double haploid.
The pollen height abortion that haplobiont produces; The pollen vigor height that double haploid produces is grown normal; Abortion to a certain degree can take place in the pollen that polyploid plant produces, and pollen grain is obviously greater than dliploid pollen.To double haploid and the pollination of high power body plant and mensuration index of affinity.
6, double haploid, high power body field shape are learned the proterties investigation
On August 5th, 2010 was sowed at the field with double haploid and the high power body seed that obtains, and obtained partial regeneration plant forms such as Fig. 4, and Fig. 5 is shown in Figure 6.
7, use
It is bright green that these plant have a blade, and the advantage of form novelty can directly be used as kind, or according to the index of affinity measurement result, utilize preparing hybrid combination between the self incompatible line, makes up with the male sterile line preparing hybrid.
Claims (3)
1. one kind obtains the method that rape belongs to A genome vegetables new germ plasm, and key step comprises:
A, according to ' Chinese cabbage subspecies * a variety of Chinese cabbage subspecies ' or ' a variety of Chinese cabbage subspecies in mutation * mutation ' combination, rape is belonged to vegetables hybridizes the acquisition seed, plantation F
1And selfing obtains F
2
B, in the greenhouse, cultivate F
1And F
2, F
1And F
2Seed through 2 ~ 4 ℃ of low temperature treatment 20 ~ 25 days, flowering stage to F
1And F
2The plant that meets breeding objective that filters out in generation carries out Isolated microspore and cultivates, need 33 ℃ of dark cultivations 1 day behind the inoculation microspore, go to 25 ℃ of incubators again and leave standstill dark cultivation 10～15 days, behind the visible embryoid of naked eyes, it is to continue dark the cultivation on 40～60 rev/mins of shaking tables that culture dish is transferred to revolution, and cultivation temperature is 25 ℃;
Above-mentioned to F
1And F
2Plant carries out Isolated microspore and cultivates, choose petal length and flower pesticide length ratio and be 0.5～0.75 open bud, after the sterilization of superclean bench inner surface, with distilled water flushing 3 times, pour the B5 medium that sucrose concentration is 130g/L into, squeeze the design of scattered small flowers and plants flower bud, make microspore dissociate out, contain the suspension of microspore with the nylon net filter in 400 order apertures, collect filtrate in 10 mL centrifuge tubes, centrifugal 3 min of 1000 rpm; Abandoning supernatant repeats 2 times; With NLN medium dilution microspore, inoculating cell density 1 * 10
5~ 2 * 10
5Individual mL
-1, to pack in the aseptic glass culture dish of 60 mm * 15 mm with every ware 5 mL microspore suspension branches, every ware adds agarose 0.5 g/L, the active carbon 10 g/L mixed liquors behind the 100 μ L high-temperature heat sterilizations; Described NLN medium does not add the NLN medium of any hormone for containing 130g/L sucrose, perhaps for containing 130g/L sucrose, adds the NLN medium of the 6-BA of 0.05 mg/L ~ 0.2 mg/L;
C, the cotyledon period embryoid is transferred to the MS medium to obtain regeneration plant;
D, regeneration plant is inoculated into the enterprising pedestrian worker's root induction of MS medium, treat that regeneration plant is taken root after, be transplanted to the greenhouse, greenhouse temperature should be 20 ℃～25 ℃ daytime, nocturnal temperature should be 5 ℃～10 ℃; Described MS medium is for containing sucrose 30g/L, agar 5.0g/L, and NAA0.1 ~ 0.2mg/L, the pH value is 5.8 ~ 6.0 MS medium.
E, after 50-70 days, regeneration plant begins to bloom, and identifies the regeneration plant ploidy, haplobiont is handled with colchicin, and it is doubled, double haploid, high power body plant keep;
F, to double haploid, high power body plant pollination self, obtain seed, measure index of affinity simultaneously;
G, plantation double haploid, high power body selfed seed according to breeding objective, are identified plant appearance character plant height, long, the leaf look of leaf, identify plant nutrition index calcium, magnesium, vitamin C, disease-resistant, resistance.
2. acquisition rape according to claim 1 belongs to the method for A genome vegetables new germ plasm, it is characterized in that: in the C step, the MS medium of regeneration induction plant is for containing sucrose 30g/L, agar 6.5g/L ~ 7.5 g/L, the pH value is 5.8 ~ 6.0, does not have the MS medium of any hormone.
3. one kind obtains the application method that rape belongs to A genome vegetables new germ plasm by the described method of claim 1: the double haploid strain is directly used as kind, or as parent and the combination of male sterile line preparing hybrid, perhaps select two self incompatible line preparing hybrid combinations.
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| CN105028203A (en) * | 2015-08-04 | 2015-11-11 | 沈阳农业大学 | Method for promoting Chinese cabbage microspore embryogeny and direct seedling development |
| CN106226137B (en) * | 2016-08-10 | 2019-04-23 | 浙江大学 | A kind of method of quick detection Brassica genus hexaploid new germ plasm ploidy |
| CN110592087A (en) * | 2019-09-24 | 2019-12-20 | 沈阳农业大学 | Application of SGR gene silencing in brassica plants |
| CN113711920B (en) * | 2021-09-26 | 2022-04-26 | 安徽农业大学 | Method for improving one-time seedling rate of microspore embryoid of non-heading Chinese cabbage |
| CN113940276B (en) * | 2021-10-20 | 2022-10-28 | 河北省农林科学院经济作物研究所 | Method for creating diploid germplasm resource by using tetraploid Chinese cabbages |
| CN114836569B (en) * | 2022-05-26 | 2024-05-17 | 沈阳农业大学 | KASP molecular marker of main effect QTL of heart branching character and application thereof |
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| 杨淑琴等.芸薹属植物游离小孢子培养研究进展.《辽宁大学学报（自然科学版）》.2005,第32卷(第1期),91-96. * |
| 王超楠.小白菜游离小孢子培养体系的构建与利用.《沈阳农业大学硕士学位论文》.2008,摘要,正文第15-16、27-28、32、37、40-42页. * |
| 陈琳等.中国小白菜小孢子离体培养和双单倍体植株再生.《中国农学通报》.2010,第26卷(第14期),78-81. * |
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