CN108077080A - A kind of in vitro directed screening method of peanut high-oil body - Google Patents
A kind of in vitro directed screening method of peanut high-oil body Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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Abstract
The present invention relates to a kind of in vitro directed screening methods of peanut high-oil body, can solve the problems, such as that peanut lacks high oily germ plasm resource, conventional breeding is difficult to cultivate high-yield high-oil peanut species.The present invention uses the high oil body of in vitro directed screening, and screening process carries out in culture medium, and a large amount of culture materials without resistance are eliminated, and can save substantial amounts of resource and cost, and can carry out mutagenesis culture and directed screening throughout the year, easy to operate.The present invention can create and screen peanut high-oil new germ plasm, widen peanut hereditary basis, overcome due to shortage height oil germ plasm resource in peanut so that the high oily breeding of new variety of high yield is difficult to the difficulty broken through.
Description
Technical field
The invention belongs to peanut breeding field more particularly to a kind of in vitro directed screening methods of peanut high-oil body.
Background technology
Peanut is the important oil crops in China, and important position is occupied in national economy.The peanut of China's production
More than 50% uses as oil expression, and general oil content more than 50% then often improves 1 hundred for high oil variety or high light wood material, oil content
Branch, can increase net profit 7%, thus cultivate high oily new peanut variety become numerous breeders main breeding objective it
One.However lack high oil variety resource in peanut, it is difficult to obtain high oily offspring, therefore the choosing of high yield high oil variety by crossbreeding
Educate an always insoluble problem.The utilization of induced-mutation technique can cause gene mutation or chromosomal variation, but be mutated
It is nondirectional, routine mutagenesis breeding can not be oriented according to breeding objective obtains required high oily mutant, after mutant
Continuous identification needs a large amount of manpowers, financial resources and material resources.It is combined using Vitro Mutation with tissue cultures and carries out in vitro directed screening height
Oil body can solve this problem.Human and material resources, financial resources can be saved, without limitation of time and space, and when shortening breeding
Between, expand spectrum of variation and improve aberration rate.The high oil body of Vitro Mutation directed screening is to obtain high oily new germ plasm, cultivate high oily new product
The effective means of kind.
Radioinduction has variation frequency height, range of variation as one of the important means of germplasm innovation and breed improvement
Greatly, offspring stablizes the features such as fast, the breeding time limit is short, and common mutation source has gamma-rays, X-ray, fast neutron, ion beam etc..I
State's peanut irradiation mutagenesis breeding is started from the 1960s, to open up new breeding method, and Recent study person are in succession to flower
Raw different method of mutagenesis are studied.Jiang Defeng etc. (nuclear agricultural science report, 2017,31,1678-1683) is utilized60Co- gamma-rays spokes
Spend No. 11 according to (irradiation metering 150Gy) peanut varieties Shandong, obtain pod character variation and crude fat content be up to 54.3% it is new
Strain, paper result illustrates that mutagenesis can improve the oil content of peanut, but can not orient acquisition, can only obtain at random, and
Some high oily mutant are easily eliminated because yield is not high.(agricultural biotechnologies, the May 25,2017,33 (5) such as Li Guan:
766-774) new peanut variety space is cultivated using Vitro Mutation and spend No. 4, disclose a kind of method for cultivating new peanut variety, the party
Mutagenesis is carried out using culture medium containing bleomycin A5 in method, afterwards using HYP directed screenings, but its method is needed in the training of domestication room
Support 3 Zhou Houzai transplanting fields, during domestication room is cultivated 3 weeks, seedling it is slow-growing and easily microbiological contamination, spend human and material resources, account for
With lab space, meanwhile, domestication room incubation influences growth.
The content of the invention
The present invention seeks to overcome above-mentioned the deficiencies in the prior art, a kind of in vitro directed screening side of peanut high-oil body is provided
Method can solve the problems, such as that peanut lacks high oily germ plasm resource, conventional breeding is difficult to cultivate high-yield high-oil peanut species.
To achieve the purpose that solve the above problems, the present invention is achieved by the following scheme：
(1) after the peanut seed of mature and plump being carried out fast neutron irradiated (irradiation dose 9Gy-20Gy), cotyledon is removed
After embryo carries out surface sterilization in superclean bench, when immersion 10~12 is small in sterile water；
(2) cotyledon Leaflet of the embryo is separated in gnotobasis as explant, is inoculated into body embryo inducing culture,
Culture 28-32 days, part explant form body embryo；The body embryo inducing culture is using MS culture mediums as minimal medium, addition 4
~12mg/L 2,4-D (2,4 dichlorophenoxyacetic acid)；Medium pH 5.8, culture room temperature is 25~27 DEG C, intensity of illumination is
When 2000~3000Lx, light application time are 12~14 small/day
(3) explant for forming body embryo is transferred in body embryo sprouting and screening and culturing medium and cultivated, carried out high oil orientation and sieve
Choosing obtains sprouting body embryo；The body embryo is sprouted and screening and culturing medium is using MS culture mediums as minimal medium, and adds 3~5m
Mol/LHYP (hydroxyproline) and 3~5mg/L BAP (6-benzyl aminopurine)；Medium pH 5.8, culture room temperature for 25~
27 DEG C, intensity of illumination be 2000~3000Lx, when light application time is 12~14 small/day；
(4) sprouting body embryo is transferred on seedling and screening and culturing medium and cultivated, carry out high oily directed screening, obtained high oily
Seedling；The seedling and screening and culturing medium add 7~9m mol/LHYP and 1~3mg/ using MS culture mediums as minimal medium
LBAP；
Specifically method is the step (4)：After sprouting body embryo cultivates 4 weeks in seedling and screening and culturing medium, transfer is extensive
Renewal cultivation 4 weeks, is then transferred on seedling and screening and culturing medium and cultivates 4 weeks on multiple culture medium, and renewal cultivation is handed over screening and culturing
For progress, until resistance seedling grows to more than 1cm, recovery media adds 1~3mg/ using MS culture mediums as minimal medium
L BAP。
Medium pH 5.8, culture room temperature is 25~27 DEG C, intensity of illumination is 2000~3000Lx, light application time 12
~14 it is small when/day.
(5) when the resistance seedling that grows up to of body embryo sprouting is grown to more than 1cm, sterile grafting can be carried out, using high oil seepage as
Scion, using peanut seedling as stock (stock is the seedling age peanut aseptic seedling of 10-12 days), grafting transplants educating in sterilizing again
Sterile culture 3~5 days in seedling matrix；Plant is in blake bottle after grafting, 25~27 DEG C of condition of culture, intensity of illumination for 2000~
When 3000Lx, light application time are 12~14 small/day.
(6) grafting hardening is directly transplanted to field after 1-2 days.It is a little that the bottleneck of blake bottle is opened during hardening, makes grafting
Seedling gradually adapts to external environment.Ridging is transplanted during transplanting, 50~60cm of row spacing, spacing in the rows 20-25cm.Grafting transplants field
Afterwards, Small plastic shed, 1 ridge of Small plastic shed span are taken with mulch, high 20~40cm is watered with water, transplants at 9 points in the morning at 10~12 days initial stages
To 4 point lid shading net in afternoon, the sun is prevented directly to shine, transplanting removes Small plastic shed after 3 weeks.
Compared with prior art, the advantages and positive effects of the present invention are：
1st, the present invention is using after fast neutron irradiated mutagenesis, the in vitro high oil body of directed screening, screening process in culture medium into
Row, a large amount of culture materials without resistance are eliminated, can save substantial amounts of resource and cost, and can carry out mutagenesis throughout the year
Culture and directed screening, it is easy to operate.The present invention can create and screen peanut high-oil new germ plasm, widen peanut hereditary basis,
Overcome due to shortage height oil germ plasm resource in peanut so that the high oily breeding of new variety of high yield is difficult to the difficulty broken through.
2nd, the present invention carries out fast neutron irradiated with ripe peanut seed, its cotyledon Leaflet then is inoculated into body embryo Fiber differentiation
Inductor embryogenesis on base.The body embryo that the explant of survival is transferred to mol/LHYP containing 4m and 3~5mg/L BAP sprouts culture
On base, oily directed screening high for the first time is carried out；The seedling training of mol/L containing 8m HYP and 1~3mg/L BAP are then transferred to after 4 weeks
It supports on base, carries out programmed screening, during postsearch screening, screen 4 weeks and recover 4 weeks, Xun Huan carries out, and can not only improve
Screening efficiency, and can effectively reduce time of the screened plant in later stage hardening.
Irradiation mutagenesis technology causes M2Plant height, flowering habit, branch amount, pod are generated in terms of biological character for plant
The characters such as character, per plant quantity substantially make a variation and separated mutant.In terms of molecular gene, the verification of SSR testing results
The validity and feasibility of the mutagenesis system, HYP directed screenings obtain high oil body offspring and the testing result of mutagenesis parent is shown
Show, between the two there are apparent gene pleiomorphism, the multiple sections of DNA molecular are interior to be occurred to repeat or lacks isostructuralism variation,
Rather than simple point mutation.
Bis- directed screening technologies of HYP obtain high oil body offspring stabilization strain oil content and are above mutagenesis parent's (oil content
49.50%), there is the high oil product system of oil content more than 55% in whole its offspring of high oil body, and part strain oil content can be with
Reach 59.63%.
3rd, the present invention high oil body that in vitro directed screening obtains on culture medium overcomes peanut tissue culture seedlings using engrafting method
The low difficulty of hardly possible, the rooting culture survival rate of taking root, by the use of seedling medium as the vernalization matrix of grafting stock, high oil seepage conduct
Scion directly transplants field after carrying out sterile grafting, can greatly save the domestication incubation time in domestication room.Direct transplant field
Between after take the Small plastic shed that mulch is made, easy to operate, moistening effect is good, is conducive to the wound healing of grafting, and can save
Human and material resources and financial resources.
Description of the drawings
The high oil body offspring phenotypic variation situation that Fig. 1 directed screenings obtain；
Fig. 2 is that flower educates No. 20 and high oil body offspring SSR electrophoretograms in the present invention；Left side is that primer pair is PM15；Right side is
Primer pair is PM297；CK：Mutagenesis parent Hua Yu 20；Other are the M3 generations of high oil body.
Specific embodiment
The present invention is described further with reference to specific embodiment, but protection scope of the present invention is not limited to this
A little embodiments.In addition any change that those of ordinary skill in the related art are the present invention, it is every without departing substantially from present inventive concept
Change or equivalent substitute be included within protection scope of the present invention.
Embodiment 1
A kind of in vitro directed screening method of peanut high-oil body of the present invention, includes the following steps：
1st, the preparation of body embryo inducing culture
(1) body embryo inducing culture is prepared, it is minimal medium to select MS culture mediums, and 2,4- are added in this MS culture medium
D-shaped adult embryonal induction culture medium.The additive amount of 2,4-D is 2~12mg/L.Body embryo inducing culture is MS+ in the present embodiment
5mg/L2,4-D+30g/L sucrose+8g/L agar；The pH of the body embryo inducing culture is adjusted to 5.8.
2nd, the preparation of body embryo Germination And Seedling and high oil body directed screening culture medium
(1) body embryo sprouting and high oily screening and culturing medium are prepared：It is minimal medium to select MS culture mediums, in this MS culture medium
Middle addition hydroxyproline and 6-benzyl aminopurine (BAP) form body embryo sprouting and high oily directed screening culture medium, hydroxyproline
Additive amount is that the additive amount of 3~5m mol/L, BAP are 3~5mg/L.Body embryo is sprouted in the present embodiment and screening and culturing medium is MS+
4m mol/L hydroxyproline+4mg/L BAP+30g/L sucrose+8g/L agar sprouts the body embryo and the pH of screening and culturing medium
It is adjusted to 5.8.
(2) seedling and high oily screening and culturing medium are prepared：It is minimal medium to select MS culture mediums, is added in this MS culture medium
Hydroxyproline and BAP is added to form seedling and high oily directed screening culture medium, the additive amount of hydroxyproline is 7~9m mol/L, BAP
Additive amount be 1~3mg/L.Seedling and screening and culturing medium are MS+8m mol/L hydroxyproline+2mg/L BAP+ in the present embodiment
The body embryo is sprouted and the pH of screening and culturing medium is adjusted to 5.8 by 30g/L sucrose+8g/L agar.
(3) restoration ecosystem culture medium is prepared：It is minimal medium to select MS culture mediums, and BAP is added in this MS culture medium
Restoration ecosystem culture medium is formed, the additive amount of BAP is 1~3mg/L.Recovery media is MS+2mg/L BAP+ in the present embodiment
The pH of the restoration ecosystem culture medium is adjusted to 5.8 by 30g/L sucrose+8g/L agar.In seedling and high oily directed screening culture medium
It after upper culture 4 weeks, is transferred on restoration ecosystem culture medium and cultivates 4 weeks, be then then transferred to seedling and high oily directed screening culture
It is cultivated 4 weeks on base, screening alternately, sprouts the seedling grown up to until body embryo and grow to more than 1cm with renewal cultivation.
By the use of the analog hydroxyproline of proline as selective agent in the present invention, the hydroxyl dried meat ammonia of limting concentration can be restrained oneself
Acid, must related gene be mutated so that be screened in object proline content and improve.Peanut is oil crops, dried meat ammonia
Acid degradation can participate in Fatty synthesis.Hydroxyproline is added in the present invention in screening and culturing medium, high oil body can be screened.BAP is one
The artificial synthesized purines plant growth regulator of kind can promote cell division, tissue differentiation, germination, lateral bud growth etc.,
It is used for inductor embryo germination and seedling in the present invention.
3rd, the establishment of hydroxyproline screening concentration
It is made an addition to using hydroxyproline as screening pressure in culture medium and is oriented the high oil body of screening.Condition of culture for 25~
27 DEG C, intensity of illumination be 2000~3000Lx, daily illumination is cultivated under conditions of being 12~14h
Explant material educates No. 20 to be widely popularized the peanut varieties of cultivation flower at present, and the seed for choosing full seed removes son
After embryo after leaf carries out surface sterilization, cotyledon Leaflet is taken as test material.
The each growth and development stage of plant is different to the tolerance of adverse circumstance.Similarly, under conditions of tissue culture each stage to inverse
The tolerance degree of border stress is also different.Adverse environmental factor is simulated with hydroxyproline, makes an addition in culture medium and is oriented the high oil of screening
Body, have studied the body embryo of peanut embryo leaflet tissue culture acquisition sprout, hydroxyproline is most in two different phases of seedling
Suitable for screening concentration.
(1) body embryo sprouts the screening of stage hydroxyproline, and the outer of body embryo is formed after cultivating for 4 week on body embryo inducing culture
Implant is transferred to body embryo and sprouts and cultivated on screening and culturing medium, and body embryo is sprouted and adds 0,2,4,6,8m in screening and culturing medium
Mol/L hydroxyprolines.After cultivating 4 weeks on the body embryo germination medium of hydroxyproline is not added with, body embryo is sprouted normal；It is adding
In the body embryo sprouting of 4m mol/L hydroxyprolines and screening and culturing medium, though body embryo seriously inhibits it without complete lethal
Growth；It is sprouted in the body embryo of addition 8m mol/L hydroxyprolines dead with the complete browning of body embryo on screening and culturing medium.Therefore, with 4m
Height oily screening concentration of the mol/L hydroxyprolines as this cultivation stage.
(2) seedling stage hydroxyproline screens, and sprouts the stage without screening in body embryo, and is normally cultivated.Embryo
Leaflet explant forms body embryo clump after being cultivated on inducing culture 4 weeks, and the explant for forming body embryo clump is transferred to and is not added with
It is further cultured on the body embryo germination medium of hydroxyproline 4 weeks, most of body embryo is sprouted.Then the body embryo clump sprouted is transferred to
It is cultivated on seedling and screening and culturing medium, 0,4,8,12m mol/L hydroxyprolines is added in seedling and screening and culturing medium.4w
Afterwards, on the culture medium of hydroxyproline is not added with, the seedling growth that body embryo sprouting grows up to is normal；In addition 4m mol/L hydroxyl dried meat ammonia
On the culture medium of acid, the seedling growth grown up to by body embryo sprouting is suppressed, but can also be grown；In addition 8m mol/L hydroxyl dried meat ammonia
The seedling growth that the seedling and screening and culturing medium upper body embryo germination of acid grow up to is heavily suppressed.Therefore, to add 8m mol/L
Height oily screening concentration of the hydroxyproline as this cultivation stage.
4th, mutagenesis conjunctive tissue culture
(1) fast neutron irradiated and cultured in vitro
Using peanut varieties flower educate the seeds of No. 20 mature and plumps for the processing of test material progress fast neutron irradiated (9~
20Gy), 9.7Gy is used in the present invention.Cotyledon is gone to remove embryo the seed after radiation treatment, be built in superclean bench
75% alcohol impregnates 20s, then impregnates 12min with 0.1% mercuric chloride, carries out surface sterilization, after rinsed with sterile water 5 times, puts
8~12h is impregnated in the blake bottle equipped with sterile water.It takes out through the soaked embryo of surface sterilization, is placed in sterile petri dish
Remove cotyledon Leaflet, be then seeded into addition 5mg/L2, on the body embryo inducing culture of 4-D, culture room temperature be 25~27 DEG C,
Intensity of illumination is 2000~3000Lx, daily illumination is cultivated under conditions of being 12~14h, induces the formation of body embryo.Culture 4
Zhou Hou, about 40% explant form body embryo, other explants only form callus or browning.And without irradiation
Seed cotyledon Leaflet explant about 80% forms body embryo.
5th, the directed screening of high oil body
The cotyledon Leaflet cultured in vitro of above-mentioned fast neutron irradiated processing is successively transferred to after 4 weeks, by the explant for forming body embryo
Promote body embryo sprouting and seedling on body embryo germination medium and seedling culture medium, and carry out the high oil body of directed screening.Using above-mentioned
Definite hydroxyproline screening concentration is oriented the high oil body of screening.4m mol/L are added first in body embryo germination medium
Hydroxyproline adds 8m mol/L hydroxyprolines in seedling culture medium, and presses and be not added with screening pressure using adding to screen afterwards
Each culture carries out alternate culture in 4 weeks, until regeneration seedling grows to more than 1cm.
Specially：It will cultivate, be transferred on body embryo sprouting and screening and culturing medium after above-mentioned fast neutron irradiated mutagenesis,
Culture room temperature is 25~27 DEG C, intensity of illumination is 2000~3000Lx, daily illumination is trained under conditions of being 12~14h
It supports, high oil directed screening and body embryo Germination And Seedling are carried out at the same time.The screening and culturing of addition 4m mol/L hydroxyprolines is selected first
Base, after cultivating 4 weeks, most of explant and body embryo are dead, only a small number of survivals.Through screening the explant with body embryo survived
It is transferred on the screening and culturing medium of addition 8m mol/L hydroxyprolines, screening and culturing has part explant and sprouting again after 4 weeks
Body embryo browning.It is then transferred to after cultivating 4 weeks on the restoration ecosystem culture medium for do not add hydroxyproline, the body embryo that part is sprouted is recovered
Growth.It is so alternately, final to obtain up to seedling afterwards again with the Screening of Media of addition 8m mol/L hydroxyprolines
Flower educates No. 20 hydroxyproline patience seedlings (high oil body).
6th, the grafting and transplanting of high oil body
(1) preparation of graft stock and sterile grafting
Select seedling medium that seedling medium is contained in blake bottle, the laggard horizontal high voltage of suitable quantity of water is added to go out as vernalization matrix
Bacterium.
The seed that flower educates No. 20 full seeds is selected to carry out surface sterilization, after impregnating 4-6 hour in sterile water, plantation
In the seedling medium of above-mentioned sterilizing, culture room temperature be 25~27 DEG C, intensity of illumination is 2000~3000Lx, daily illumination
To be cultivated under conditions of 12~14h, stock germination, hypocotyl elongation after 11 days can be grafted.
The body embryo that above-mentioned flower is educated to No. 20 mutagenesis and directed screening acquisition sprouts the seedling grown up to as scion, is cut into " ∨ "
Font, the flower of sterile vernalization educate No. 20 seedlings as stock, cut off cotyledonary node above section, indulged in hypocotyl middle part
Cut, scion afterwards insertion stock in, graft union is sealed with sealed membrane, dehydration is prevented, is also in close contact interface, make stock and
The microtubule system of scion is connected.
(2) grafting rooting culture
Above-mentioned grafting is transplanted again in the seedling medium of sterilizing, is 25~27 DEG C, intensity of illumination in culture room temperature
It is cultivated 4 days under conditions of being 12~14h for 2000~3000Lx, daily illumination.It opens bottle cap to carry out a little after taming 2 days, directly
Meet transplanting field, the plant of grafting ridging, 50~60cm of row spacing, spacing in the rows 20-25cm.Behind grafting transplanting field, taken with mulch
Small plastic shed per 1, ridge shed, is watered with water, transplants 4 point lid shading net at 9 points in the morning to afternoon 10~12 day initial stage, prevents that the sun is straight
It shines.Small plastic shed is removed after transplanting 3 weeks, carries out field management by normal method, grafting normal growth is bloomed and result.
7th, high oil body progeny variation and separation
It transplants the high oil seepage of grafting (body) in crop field to harvest by single plant, next year kind is into plant, with mutagenesis parent Hua Yu 20
As control.Flower educates No. 20 38~42cm of plant height, and sparse branching, branch amount 9~11, flower habit is continuously blooms, the common shape of pod,
Kernel seed coat colour pink.High oil body offspring is in plant height, flowering habit, branch amount, per plant number, pod shape, kernel seed coat colour
Etc. various aspects show significantly variation and separation.
No. b high oil body offspring per plant number increases, and branch amount becomes more, b-1 and b-2 branch amount 16-17 items, such as Fig. 1-b
It is shown.
No. d high oil body offspring branch amount, pod shape, kernel seed coat colour substantially make a variation and separate, and d-1 branch amounts increase, pod
The mutation of fruit shape shape is separated into beading shape, and kernel seed coat colour sports aubergine, and d-2 branch amounts also increase, but pod shape is general
Logical shape, kernel seed coat colour are pink, and d-3 branch amounts are unchanged, but pod contracting is hung profound, and shape sports Pear-Shaped, such as Fig. 1-d
It is shown.
Substantially variation and separation, e-2 per plant numbers tail off for No. e high oil body offspring pod shape, per plant number, pod
Become smaller, pod contracting in part is hung depth, and shape sports Pear-Shaped, and e-3 per plants number becomes more, and part pod contracts unobvious of hanging, shape
Shape is silk cocoon shape, and flower educates 20 contractings and hangs substantially, and shape is common shape, as shown in Fig. 1-e.
Substantially variation and the separation of No. f high oil body offspring pod shape, per plant number, f-1 per plant numbers are few, f-2
The contracting of single pod is hung depth, and shape becomes Pear-Shaped, as shown in Fig. 1-f.
8th, high oil body offspring oil content detection
Peanut is one of oil crops, and general kind oil content 50% or so, oil content reaches more than 55% and is then set to height
Oil product kind or high light wood material.Oil content often improves 1 percentage point, and net profit can increase by 7%
The above-mentioned high oil body that the acquisition of in vitro directed screening is combined using Vitro Mutation is carried out after being selfed homozygosis, lines progeny seed
Benevolence oil content is measured using near-infrared method, and all high oil body offspring oil contents (50.01%~60.03%) are above mutagenesis
Parent Hua Yu No. 20 (oil contents 49.50%).
The high strain of 30 oil contents of selection handed Ministry of Agriculture's oil plant and quality of item prison over to respectively at 2014 and 2015
Verification test center is superintended and directed to test, using mutagenesis parent Hua Yu 20 as control, inspection result is as shown in table 1,
As can be seen from Table 1, No. 20 oil contents of mutagenesis parent Hua Yu are 49.50%, detected 30 high oil body offsprings
In strain, 28 oil contents reach high oily (more than 55%) standard, wherein 6 oil contents reach more than 59%, 10 oil contents
In 58%~59%, up to 59.63%.
1 high oil body offspring oil content of table
| Number | Oil content (%) |
| Flower educates 20 | 49.50 |
| 1 | 57.56 |
| 2 | 59.48 |
| 3 | 57.23 |
| 4 | 58.45 |
| 5 | 56.68 |
| 6 | 57.23 |
| 7 | 57.73 |
| 8 | 58.34 |
| 9 | 56.47 |
| 10 | 58.06 |
| 11 | 56.93 |
| 12 | 58.02 |
| 13 | 59.52 |
| 14 | 57.22 |
| 15 | 56.10 |
| 16 | 58.17 |
| 17 | 58.49 |
| 18 | 56.52 |
| 19 | 58.40 |
| 20 | 58.83 |
| 21 | 56.77 |
| 22 | 54.28 |
| 23 | 54.56 |
| 24 | 58.65 |
| 25 | 59.38 |
| 26 | 58.96 |
| 27 | 57.88 |
| 28 | 59.63 |
| 29 | 59.50 |
| 30 | 59.06 |
9th, the SSR detections of high oil body
Offspring's (from different high oil bodies) of 13 high oil bodies is randomly choosed, blade total DNA is extracted, utilizes SSR primers
To being detected analysis DNA variations.The amplified production for wherein having 7 pairs of primers can disclose mutant material and mutagenesis parent Hua Yu 20
Polymorphism between number, and in 13 detected materials 12 show 2 or more site mutations, as shown in table 2.By table 2
As can be seen that with mutagenesis parent Hua Yu No. 20 compare, the specific manifestation form of SSR marker polymorphism has:Amplified fragments number
It reduces and (is labeled as A)；Amplified fragments number increases and (is labeled as B)；Expanding fragment length difference (is labeled as C).A, B, C these three
Form proportion is 20.69%, 27.59%, 51.72% respectively.Show that high oil mutation is the strain since gene is mutated
Height, flowering habit, branch amount, per plant number, pod shape, kernel seed coat colour etc. are since gene is undergone mutation.Same height
It is since high oily mutator is happened on wherein one of homologue that separation, which occurs, for oil body offspring oil content, is being selfed
Cheng Zhong, the Gene Isolation and homozygosis carried due to homologue so that oil content is different between Sister Lines.Fig. 2 is c-1, k-
12nd, the electrophoretogram of d-2, j-5 and l-12.
No. 20 SSR Polymorphism Analysis of 2 high oil body of table and mutagenesis parent Hua Yu
Note：A:Amplified fragments number is reduced；B：Amplified fragments number increases；C：Expanding fragment length difference
Specific primer is as follows：
The present invention irradiates conjunctive tissue culture, the indirect selection pressure selected using hydroxyproline as high oil screen using fast seed
Power, the high oil body of acquisition directly transplant field through sterile grafting, obtain the seed of normal mature.It is sent out in high oil body offspring phenotype
Raw apparent variation and separation make high oil body offspring homozygous, oil content, high oil body offspring are measured using near-infrared method by selfing
Oil content is above mutagenesis parent (oil content 49.50%), is examined through Ministry of Agriculture's oil plant and quality of item supervision and inspection center
Test oil content, in detected 30 high oil body lines progenies, 28 oil contents reach high oily (more than 55%) standard, wherein 6
A oil content reaches more than 59%, and 10 oil contents are in 58%~59%, up to 59.63%.Utilize the detection point of SSR methods
DNA variations are analysed, 12 detect that 2 or more sites are different in offspring's (coming from different high oil bodies) of tested 13 high oil bodies
In mutagenesis parent, the oil content raising for illustrating these materials is due to gene mutation；In addition the variation of phenotype, yield
Raising is also due to the reason for gene mutation.
Sequence table
<110>Qingdao Agricultural University
<120>A kind of in vitro directed screening method of peanut high-oil body
<130> 2018
<141> 2018-02-07
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence (Arachis hypogaea Linn.)
<400> 1
atcaccatca gaacgatccc 20
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence (Arachis hypogaea Linn.)
<400> 2
tttgtagcct tctggcgagt 20
<210> 3
<211> 22
<212> DNA
<213>Artificial sequence (Arachis hypogaea Linn.)
<400> 3
aagaagaggc ttggttggga tg 22
<210> 4
<211> 23
<212> DNA
<213>Artificial sequence (Arachis hypogaea Linn.)
<400> 4
caagggtgga gagataatgc aca 23
<210> 5
<211> 20
<212> DNA
<213>Artificial sequence (Arachis hypogaea Linn.)
<400> 5
atgcacctgc aagtgaagag 20
<210> 6
<211> 20
<212> DNA
<213>Artificial sequence (Arachis hypogaea Linn.)
<400> 6
tcaaggatgc agcaagacac 20
<210> 7
<211> 25
<212> DNA
<213>Artificial sequence (Arachis hypogaea Linn.)
<400> 7
ccttttctaa cacattcaca catga 25
<210> 8
<211> 19
<212> DNA
<213>Artificial sequence (Arachis hypogaea Linn.)
<400> 8
ggctcccttc gatgatgac 19
<210> 9
<211> 20
<212> DNA
<213>Artificial sequence (Arachis hypogaea Linn.)
<400> 9
tgggcctaaa cccaacctat 20
<210> 10
<211> 20
<212> DNA
<213>Artificial sequence (Arachis hypogaea Linn.)
<400> 10
ccacaaacag tgcagcaatc 20
<210> 11
<211> 20
<212> DNA
<213>Artificial sequence (Arachis hypogaea Linn.)
<400> 11
ctttcttccc ccttgaacct 20
<210> 12
<211> 25
<212> DNA
<213>Artificial sequence (Arachis hypogaea Linn.)
<400> 12
gatcaagtga aaatgttagt ataag 25
<210> 13
<211> 20
<212> DNA
<213>Artificial sequence (Arachis hypogaea Linn.)
<400> 13
gggcttcact gcttttgatt 20
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<211> 20
<212> DNA
<213>Artificial sequence (Arachis hypogaea Linn.)
<400> 14
tgcgacttct gagaggacaa 20
Claims (10)
1. a kind of in vitro directed screening method of peanut high-oil body, it is characterised in that it includes the following steps：
(1) by after mutagenic treatment go cotyledon embryo the explant survived after Fiber differentiation be transferred to body embryo sprout and
It is cultivated on screening and culturing medium, carries out high oily directed screening, obtain sprouting body embryo；The body embryo is sprouted and screening and culturing medium is trained with MS
It is minimal medium to support base, and adds 3~5m mol/L HYP and 3~5mg/L BAP；
(2) sprouting body embryo is transferred on seedling and screening and culturing medium and cultivated, carried out high oily directed screening, obtain high oil seepage；Institute
Seedling and screening and culturing medium are stated using MS culture mediums as minimal medium, and adds 7~9m mol/LHYP and 1~3mg/L BAP；
(3) when the resistance seedling that body embryo sprouting grows up to is grown to more than 1cm, sterile grafting can be carried out, using high oil seepage as scion,
Using peanut seedling as stock, grafting transplants sterile culture 3~5 days in the seedling medium of sterilizing again；
(4) grafting hardening is directly transplanted to field after 1-2 days.
2. the in vitro directed screening method of peanut high-oil body according to claim 1, it is characterised in that it includes following step
Suddenly：
(1) after the peanut seed of mature and plump being carried out fast neutron irradiated, the embryo of cotyledon carry out table in superclean bench is removed
Face sterilizes；
(2) cotyledon Leaflet of the embryo is separated in gnotobasis as explant, is inoculated into body embryo inducing culture, cultivates
28-32 days, part explant formed body embryo；The body embryo inducing culture using MS culture mediums as minimal medium, addition 4~
12mg/L 2,4-D；
(3) explant for forming body embryo is transferred in body embryo sprouting and screening and culturing medium and cultivated, carried out high oily directed screening, obtain
To sprouting body embryo；The body embryo is sprouted and screening and culturing medium is using MS culture mediums as minimal medium, and adds 3~5m mol/L
HYP and 3~5mg/L BAP；
(4) sprouting body embryo is transferred on seedling and screening and culturing medium and cultivated, carried out high oily directed screening, obtain high oil seepage；Institute
Seedling and screening and culturing medium are stated using MS culture mediums as minimal medium, and adds 7~9m mol/LHYP and 1~3mg/L BAP；
(5) when the resistance seedling that body embryo sprouting grows up to is grown to more than 1cm, sterile grafting can be carried out, using high oil seepage as scion,
Using peanut seedling as stock, grafting transplants sterile culture 3~5 days in the seedling medium of sterilizing again；
(6) grafting hardening is directly transplanted to field after 1-2 days.
3. a kind of in vitro directed screening method of peanut high-oil body according to claim 1, it is characterised in that：The step
(1) fast neutron irradiated dosage is 9Gy-20Gy in.
4. a kind of in vitro directed screening method of peanut high-oil body according to claim 1, it is characterised in that：The step
(1) after going the embryo surface sterilization of cotyledon in, when immersion 10~12 is small in sterile water.
5. a kind of in vitro directed screening method of peanut high-oil body according to claim 1, it is characterised in that：The step
(4) after sprouting body embryo cultivates 4 weeks in seedling and screening and culturing medium in, renewal cultivation 4 weeks on recovery media is shifted, are retransferred
Cultivated 4 weeks on to seedling and screening and culturing medium, renewal cultivation and screening and culturing alternately, until resistance seedling grow to 1cm with
On, recovery media adds 1~3mg/L BAP using MS culture mediums as minimal medium.
6. a kind of in vitro directed screening method of peanut high-oil body according to claim 1, it is characterised in that：The step
(2), condition of culture is in (3) and (4)：Medium pH 5.8, culture room temperature is 25~27 DEG C, intensity of illumination be 2000~
When 3000Lx, light application time are 12~14 small/day.
7. a kind of in vitro directed screening method of peanut high-oil body according to claim 1, it is characterised in that：The step
(5) stock is the seedling age peanut aseptic seedling of 10-12 days in.
8. a kind of in vitro directed screening method of peanut high-oil body according to claim 1, it is characterised in that：The step
(5) plant is in blake bottle after grafting in, and 25~27 DEG C of condition of culture, intensity of illumination are 2000~3000Lx, light application time is
12~14 it is small when/day.
9. a kind of in vitro directed screening method of peanut high-oil body according to claim 7, it is characterised in that：The step
(6) bottleneck for opening blake bottle in during hardening is a little.
10. a kind of in vitro directed screening method of peanut high-oil body according to claim 1, it is characterised in that：The step
Suddenly ridging is transplanted during transplanting in (6), 50~60cm of row spacing, spacing in the rows 20-25cm；In the step (6) behind grafting transplanting field,
Small plastic shed, 1 ridge of Small plastic shed span are taken with mulch, high 20~40cm is watered with water, and arrive in the at 9 points in the morning at 10~12 days transplanting initial stages
Afternoon, 4 point lid shading net, prevented the sun directly to shine, and transplanting removes Small plastic shed after 3 weeks.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810125227.7A CN108077080A (en) | 2018-02-08 | 2018-02-08 | A kind of in vitro directed screening method of peanut high-oil body |
| CN202010765852.5A CN111837957A (en) | 2018-02-08 | 2018-02-08 | Culture medium for in-vitro directional screening of high-oil bodies of peanuts and screening method |
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| CN201810125227.7A CN108077080A (en) | 2018-02-08 | 2018-02-08 | A kind of in vitro directed screening method of peanut high-oil body |
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| CN201810125227.7A Pending CN108077080A (en) | 2018-02-08 | 2018-02-08 | A kind of in vitro directed screening method of peanut high-oil body |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109156352A (en) * | 2018-10-09 | 2019-01-08 | 青岛农业大学 | One cultivate peanut mutant rapid screening method and application |
| CN109430063A (en) * | 2018-02-09 | 2019-03-08 | 青岛农业大学 | A kind of directed screening method of floorboard with high oil content peanut |
| CN109479721A (en) * | 2018-12-29 | 2019-03-19 | 青岛农业大学 | A kind of peanut plant regeneration method |
| CN117016385A (en) * | 2023-08-24 | 2023-11-10 | 安徽省农业科学院作物研究所 | Peanut transverse nitrogen ion beam implantation mutagenesis method |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102792892A (en) * | 2012-08-21 | 2012-11-28 | 青岛农业大学 | Method for directional screening of resistance body by peanut in-vitro mutation |
-
2018
- 2018-02-08 CN CN201810125227.7A patent/CN108077080A/en active Pending
- 2018-02-08 CN CN202010765852.5A patent/CN111837957A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109430063A (en) * | 2018-02-09 | 2019-03-08 | 青岛农业大学 | A kind of directed screening method of floorboard with high oil content peanut |
| CN109156352A (en) * | 2018-10-09 | 2019-01-08 | 青岛农业大学 | One cultivate peanut mutant rapid screening method and application |
| CN109479721A (en) * | 2018-12-29 | 2019-03-19 | 青岛农业大学 | A kind of peanut plant regeneration method |
| CN117016385A (en) * | 2023-08-24 | 2023-11-10 | 安徽省农业科学院作物研究所 | Peanut transverse nitrogen ion beam implantation mutagenesis method |
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| Publication number | Publication date |
|---|---|
| CN111837957A (en) | 2020-10-30 |
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