CN102792892A - Method for directional screening of resistance body by peanut in-vitro mutation - Google Patents
Method for directional screening of resistance body by peanut in-vitro mutation Download PDFInfo
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Abstract
The invention provides a method for directional screening of a resistance body by peanut in-vitro mutation, which comprises the following main steps: disinfecting surfaces of peanut seed embryos, rinsing and soaking with sterile water; separating embryonic leaflets, inoculating the embryonic leaflets into a somatic embryo induction medium for mutation culture; transferring explants forming the somatic embryos onto a somatic embryo germination medium, adding hydroxyproline into the medium as screening stress, performing directional screening of resistance bodies, grafting and transplanting the obtained resistance seedlings into field, performing routine field management. Most progeny plants of the resistance bodies obtained in the invention are subject to obvious variation and separation; the drought resistance is obvious, and the SOD enzyme activity and the POD enzyme activity are obviously improved; the mutation material of the invention is not limited by seasons; anti-adversity mutants are directionally screened by in-vitro mutation combined with tissue culture, which saves a lot of manpower, material resources, and financial resources; the mutant regeneration is realized through an embryogenetic approach, and somatic cell embryos are derived from one cell, which avoid mutant mosaicism.
Description
Technical field
The invention belongs to the peanut breeding field, relate in particular to the method for mutagenesis directed screening resistance body of exsomatizing of cultivating peanut.
Background technology
Along with the continuous deterioration of environment, environment stress such as arid, the saline and alkaline problem that become international, the crop new varieties of cultivating multiple resistance have become one of main breeding objective of vast breeding man.Peanut is one of the important oil crop of China and economic crops, yet lacks high resistant variety resource in the peanut cultivation kind, and the seed selection of anti-adversity is an insoluble problem always.The utilization of induced-mutation technique can produce the non-existent new proterties of natural world, newtype, utilizes mutagenesis to combine with tissue culture and carries out the directed screening mutant, can save human and material resources, financial resources, shortening the breeding cycle.Therefore, exsomatizing that mutagenesis combines with tissue culture is the effective means of acquisition new germ plasm.
Utilize mutagenesis to combine to create new germ plasm resource research and on various plants, carry out with Plant Tissue Breeding; Obtained various good mutant at aspects such as disease resistance, resistance, ripe property, output, qualities, the mutant that has is directly used or has been bred new varieties as parent material.In recent years, only a few is reported the stripped mutagenesis research of relevant peanut, but the degeneration-resistant mutant of mutagenesis directed screening that exsomatizes does not appear in the newspapers at present.
Summary of the invention
The invention provides the method for the degeneration-resistant mutant of mutagenesis directed screening of exsomatizing of cultivating peanut; The present invention carries out directed screening resistance body through the mutagenesis of exsomatizing; Can create the degeneration-resistant new germ plasm of peanut, overcome the peanut hereditary basis narrow, lack high resistant variety resource, cause the peanut breeding for stress tolerance to be difficult to obtain the difficulty of breakthrough; And can save a large amount of human and material resources, financial resources, shortening the breeding cycle.
For reaching the purpose that addresses the above problem, the present invention adopts following technical scheme to be achieved:
The exsomatize method of mutagenesis directed screening resistance body that one cultivates peanut, it comprises the steps:
(1) go the embryo behind the cotyledon to carry out surface sterilization ripe peanut seed;
(2) the embryo leaflet of the said embryo of separation is an explant under aseptic condition, and the embryo leaflet is inoculated in the body embryonal induction medium, carries out mutagenesis and cultivates 25～30d, few part explant organizator embryo; Said body embryonal induction medium is with MSB
5Be minimal medium, and add 3～4mg/L bleomycin A5 and 5～10mg/L 2,4-D;
(3) explant of organizator embryo is transferred to cultivate on the body embryo germination medium obtains the resistance regrowth, said body embryo germination medium is with MSB
5Be minimal medium, and add 4～8mg/L hydroxyproline and 4mg/L BAP;
(4) when the resistance regrowth grows to 2～3cm, downcut from base portion, graft on the plumular axis of peanut seedling of axenic germination, behind the aseptic culture 1-2d, transplant acclimation shaking culture in seedling medium, after be transplanted to the field and cultivate.
Further improvement to technique scheme: adopt in the said step (3) to add screening and press and do not add screening and press and respectively cultivate 4w and replace cultivation.
Further improvement to technique scheme: the degeneration-resistant screening concentration of hydroxyproline in body embryo germination stage is 4mg/L in the said step (3), and the degeneration-resistant screening concentration of hydroxyproline is 8mg/L during successive transfer culture.
Further improvement to technique scheme: alcohol-pickled 15～25s of 75% is adopted in the surface sterilization of peanut embryo in the said step (1), with 0.1% mercuric chloride immersion, then with after the rinsed with sterile water, places sterile water to soak 12～16h again.
Further improvement to technique scheme: condition of culture is in said step (2) and (3): temperature is that 24～26 ℃, intensity of illumination are that 2000～3000Lx, light application time are 10-15h/d, medium transfer to pH 5.8.
Further improvement to technique scheme: said body embryonal induction medium is MSB
5+ 5 mg/L 2,4-D+3mg/L PYM+30g/L sucrose+8g/L agar; Said body embryo germination medium is MSB
5+ 4 mg/L BAP+30g/L sucrose+8g/L agar+4mg/L hydroxyproline.
Further improvement to technique scheme: the peanut aseptic seedling with seedling age 9-12d in the said step (4) is a stock, and the resistance regrowth is that aseptic grafting is carried out in scion.
Further improvement to technique scheme: the condition of said acclimation shaking culture is 22 ~ 26 ℃, and 18～25d is cultivated in the domestication chamber.
Advantage of the present invention and good effect are:
1, the present invention is an explant with the embryo leaflet of peanut mature seed embryo, in minimal medium, adds 3～4mg/L bleomycin A5 and 5～10mg/L 2, and 4-D (Benzene Chloride fluoroacetic acid class weed killer herbicide) carries out mutagenic treatment.The explant of organizator embryo is transferred on the medium that adds 4～8mg/L hydroxyproline and 4mg BAP (6-benzyl aminopurine) again and is impelled body embryo germination Cheng Miao, carries out directed screening resistance body simultaneously.The present invention makes an addition in the medium as degeneration-resistant screening pressure with hydroxyproline (HYP), the screening concentration that each cultivation stage is suitable is studied, and obtained the resistance body.The hydroxyproline resistance seedling that obtains is through between the transplant field of grafting domestication back, and mature seed next year of acquisition is sowed the field, and the parent compares with mutagenesis, most of resistance seedling offspring plant showed remarkable variation with separate, and compare with the mutagenesis parent and to show evident difference; Compare with the mutagenesis parent under the arid treatment conditions, the resistance body surface reveals tangible drought resistance, and SOD (superoxide dismutase) is active and POD (peroxidase) activity obviously raises.
2, the present invention can not receive season limit with the peanut mature seed as mutant materials, can carry out mutagenic treatment and directed screening throughout the year.
3, utilize the mutagenesis conjunctive tissue that exsomatizes to cultivate the degeneration-resistant mutant of directed screening, screening process is carried out in medium, and the culture materials that does not have resistance in a large number is eliminated, and can save great amount of manpower and material resources, financial resources; The regeneration of mutant is through embryo's generation approach, and somatic embryo originates from a cell, can avoid the mosaic of mutant.
The present invention lays a good foundation for the new breeding approach of developing peanut, and the drought resisting resistance body of acquisition can directly or indirectly be used for peanut breeding.
After advantages embodiment of the present invention, other characteristics of the present invention and advantage will become clearer.
Description of drawings
Fig. 1 is illustrated in peanut embryo leaflet formation regular embryo on the inducing culture that does not add HYP.
Fig. 2 shows the influence of variable concentrations HYP to the growth of peanut embryo leaflet, A: the body embryonal induction medium that adds 2 mg/L HYP; B: the body embryonal induction medium that adds 4 mg/L HYP; C: the body embryonal induction medium that adds 6mg/L HYP; D: the body embryonal induction medium that adds 8 mg/L HYP.
Fig. 3 shows among the present invention HYP to the influence of body embryo germination, A: the body embryo germination medium that does not add HYP; B: the body embryo germination medium that adds 4 mg/L HYP; C: the body embryo germination medium that adds 8 mg/L HYP.
HYP was to the influence of body embryo germination Cheng Miao, A: the body embryo germination medium that does not add HYP when Fig. 4 showed successive transfer culture among the present invention; B: the body embryo germination medium that adds 8 mg/L HYP.
Fig. 5 be among the present invention flower to educate exsomatize mutagenesis, directed screening and resistance seedlings of No. 20 embryo leaflets solid; A: the body embryo that on the inducing culture that adds 3mg/L PYM, forms; B: the resistance seedling that successively screening obtains on the body embryo germination medium that adds 4mg/L HYP and 8mg/L HYP; C: the resistance seedling of performance variation; D: the resistance seedling is solid.
Fig. 6 is mutagenesis parent and mutant M among the present invention
2Representative type and separation case thereof; A: No. 20, mutagenesis parent Hua Yu; The B:43 strain is that the plant height change is short; The C:42 strain is that stem branch color, plant type, flowering habit morph; The D:33 strain is that plant height obviously separates.
Fig. 7 is after arid is handled among the present invention, mutagenesis parent and mutant M
2The upgrowth situation in generation; A: No. 20, mutagenesis parent Hua Yu; B:41 strain system; C:42 strain system.
Fig. 8 is anti-HYP resistance body M among the present invention
2The SOD in generation is active.
Fig. 9 is anti-HYP resistance body M among the present invention
2The POD in generation is active.
Embodiment
Below in conjunction with accompanying drawing and embodiment technical scheme of the present invention is done further detailed explanation.
Embodiment 1
The method of the stripped mutagenesis directed screening resistance body of peanut according to the invention comprises the steps:
1. the preparation of medium
(1) preparation mutagenesis and body embryonal induction medium: choose MSB
5(MS mineral salt+B
5Organic principle) is minimal medium, at this MSB
5Add bleomycin A5 and 2 in the medium, 4-D forms mutagenesis and body embryonal induction medium.The addition of bleomycin A5 is 3～4mg/L, 2, and the addition of 4-D is 5～10mg/L.Body embryonal induction medium is MSB in the present embodiment
5+ 5 mg/L 2,4-D+3 mg/L bleomycin A5+30g/L sucrose+8g/L agar; The pH of said body embryonal induction medium is transferred to 5.8, is that 24～26 ℃, intensity of illumination are that 2500～3000Lx, illumination every day are to cultivate under the condition of 10～15h in culturing room's temperature.Mutagenesis and body embryonal induction carry out simultaneously.
Bleomycin A5 (PYM) is a kind of antibiotic, belongs to a type of bleomycin, is the A5 component of bleomycin, and it has characteristics such as safe, efficient, that mutagenic frequency is high, scope is big as mutagen.2,4-D is a kind of Benzene Chloride fluoroacetic acid class weed killer herbicide, also can be used as plant growth regulating, is to be used for the growth hormone commonly used that evoked callus forms, and is used for the inductor embryogenesis in the present invention.
(2) degeneration-resistant screening of preparation and body embryo germination medium: choose MSB
5(MS mineral salt+B
5Organic principle) is minimal medium, at this MSB
5Add hydroxyproline (HYP) and 6-benzyl aminopurine (BAP) in the medium and form degeneration-resistant directed screening and body embryo germination medium, the addition of hydroxyproline is 4～8mg/L, and the addition of BAP is 4mg/L.Body embryo germination medium is MSB in the present embodiment
5+ 4～8mg/L HYP+4 mg/L BAP+30g/L sucrose+8g/L agar; The pH of said body embryo germination medium is transferred to 5.8, is that 24～26 ℃, intensity of illumination are that 2500～3000Lx, illumination every day are to cultivate under the condition of 10～15h in culturing room's temperature.Directed screening and body embryo germination carry out
simultaneously.
2. HYP screening concentration confirms
Make an addition to as degeneration-resistant screening pressure with HYP and to carry out directed screening resistance body in the medium.
At first establish suitable HYP critical concentration, test is carried out at 3 different times cultivating respectively: handle I, add 0,2,4,6,8 mg/L HYP in the inducing culture; Handle II, transfer on the body embryo germination medium and cultivate not adding the body embryo clump that forms on the inducing culture of HYP, add 0,4,6,8,10 mg/L HYP in the body embryo germination medium; Handle III, the sprouting body embryo clump that obtains on the medium of HYP is not all added in preceding 2 cultivations, transfers on the fresh body embryo germination medium and carries out successive transfer culture, adds 8,10,12 mg/L HYP in the medium.Each stage is cultivated viewing test result behind the 4w respectively.
Peanut is different to the tolerance of adverse circumstance each period of growing.In like manner, each stage is also different to the tolerance degree of environment stress under the conditions of tissue culture.With HYP simulation adverse environmental factor, make an addition to and carry out directed screening resistance body in the medium, studied the peanut embryo leaflet tissue and cultivated the best screening concentration through HYP in three different phases of somatic embryo generation approach regeneration plant.
Handle I, the embryo leaflet is seeded in adds variable concentrations HYP (0,2,4; 6,8 mg/L) on the inducing culture, the result behind the cultivation 4w is as illustrated in fig. 1 and 2, as can be seen from the figure; The formation of HYP severe inhibition body embryo is except that contrast (shown in Figure 1), on the inducing culture of above-mentioned interpolation variable concentrations HYP; HYP concentration height (shown in Fig. 2 A, B, C and the D) no matter, the little leaf explant of embryo all forms loose callus or brownization, does not all observe the formation of body embryo on all medium.Therefore judge that the body embryonal induction stage is inappropriate for directed screening.
Handle II, do not add cultivate 4w on the inducing culture of HYP after the explant of organizator embryo, transfer on the body embryo germination medium that adds HYP (0,4,6,8,10 mg/L) and cultivate.Experimental result is as shown in Figure 3 after cultivating 4w, as can be seen from Figure 3, is not adding on the body embryo germination medium of HYP (Fig. 3 A), and the body embryo germination is normal; On the body embryo germination medium that adds 4 mg/L HYP (Fig. 3 B), though the body embryo does not have complete lethal, but its growth of severe inhibition; Complete brownization of (Fig. 3 C) body embryo is dead on the body embryo germination medium that adds 8 mg/L HYP.Therefore, with the degeneration-resistant screening concentration of 4 mg/L HYP as this cultivation stage.
Handle III, the processing stage of preceding 2, all do not carry out degeneration-resistant screening, and cultivate normally.The little leaf explant of embryo is organizator embryo clump after cultivating 4w on the inducing culture, body embryo clump is transferred on the body embryo germination medium cultivated 4w again, most of body embryo germination.Then the body embryo clump that sprouts is transferred to and carried out successive transfer culture on the fresh medium, add 0,8,10,12 mg/L HYP in the medium.Experimental result is as shown in Figure 4 behind the 4w, as can be seen from Figure 4, is not adding (shown in Fig. 4 A) on the medium of HYP, and the seedling growth that the body embryo germination grows up to is normal; On the medium that adds 8 mg/L HYP (shown in Fig. 4 B), the seedling growth that is grown up to by the body embryo germination is suppressed.Therefore, this stage HYP drought resisting screening concentration is confirmed as 8 mg/L.
3. bleomycin A5 mutagenic treatment
Educating No. 20 seed embryo leaflets with flower is the mutagenesis culture materials,, makes an addition to and carries out mutagenic treatment in the medium as mutagen with bleomycin A5 (PYM).
Choose full flower and educate No. 20 seeds and remove cotyledon, embryo is placed 75% alcohol-pickled 20s, soak 10min with 0.1% mercuric chloride again, carry out surface sterilization, after rinsed with sterile water 5 times, place the wide-mouth bottle that sterile water is housed to soak 12～16h.Take out the embryo that soaks, place sterile petri dish to separate the embryo leaflet, be inoculated into then on the inducing culture that adds 3mg/L PYM and cultivate 4w, carry out the formation of mutagenic treatment and inductor embryo.
4.HYP the directed screening of resistance seedling
Flower is educated No. 20 seed embryo leaflets cultivations and on the inducing culture that adds 3mg/L PYM, is carried out mutagenic treatment.After cultivating 10d, explant begins to form the milky callus, cultivates 4w, and the part callus is brownization or form the vitrifying callus gradually, and other has the part can the organizator embryo, shown in Fig. 5 A.
After 4w is cultivated in mutagenesis, the explant of organizator embryo transferred on the body embryo germination medium impel the body embryo germination, and carry out directed screening resistance body.Utilize above-mentioned definite HYP screening concentration, carry out embryo leaflet explant mutagenic treatment and directed screening resistance body.At first in body embryo germination medium, add 4 mg/L HYP, be increased to 8 mg/L during successive transfer culture.For the culture materials of avoiding screening was grown slow or brown stain, when on body embryo germination medium, screening, adopt to add screening and press and do not add screening and press and respectively cultivate 4w and replace cultivation.Contain on the medium of HYP cultivate 4w after, most of explant and body embryo are dead, and the minority survival is only arranged; The explant of the organizator embryo of surviving forwards on the body embryo germination medium that does not contain the screening pressure; Slowly recover growth, the part of contact medium forms a large amount of green fine and close callus, goes on the screening culture medium again behind the 4w; So circulate repeatedly, until emerge (Fig. 5 B).Finally obtain a small amount of flower and educated the resistance seedling No. 20.
5. the grafting of anti-HYP regrowth and transplanting
Educating No. 20 regrowths with the flower of above-mentioned screening acquisition is that scion, colored aseptic seedling of educating No. 23 are stock; At [Hao Shijun etc. such as super-clean bench internal reference Hao generation persons of outstanding talent; The research [J] of peanut tissue cultivating seedling graft technology. Qingdao Agricultural University's journal, 27 (2): 110-113] method carry out aseptic grafting, domestication is transplanted and management.Carried out the grafting of resistance seedling in 2011, after domestication, transplant in Qingdao Agricultural University experimental field, growth and development stage is observed and is put down in writing, and individual plant results pod is divided in ripe back.Seed with the individual plant results in 2012, part is by (the M of strain system
2For) plant in plastic tunnel and carry out the arid processing; Remainder is by (the M of strain system
2Generation) be seeded in Qingdao Agricultural University experimental field, seedling stage and growth and development stage are observed record character variation and separation case.
When HYP resistance regrowth grows to 2cm, just can carry out aseptic grafting.Educating No. 23 aseptic seedling with the flower of the about 10d of seedling age is stock, and the resistance regrowth is that aseptic grafting is carried out in scion.Grafting is transplanted after taming in the experimental field, observes and finds that No. 42 regrowth promptly shows variation the present age, and stem branch color becomes purple (Fig. 5 C), and the mutagenesis parent is green.Grafting carries out field management by routine, and individual plant results pod is pressed in ripe back, has 13 strain resistance seedlings and has obtained M
2For seed (Fig. 5 D).
6. resistance seedling M
2In generation, separate and the variation situation
M with results
2For seed, next year the part seed be seeded in the land for growing field crops by strain system, compare for No. 20 with mutagenesis parent Hua Yu.The growth and development stage observed result shows; In 13 strain systems (31 ~ No. 43); Except that No. 32 nothing obviously makes a variation; It is different with the mutagenesis parent that other 12 strains tie up to aspects such as plant height, stem branch color, plant type, flowering habit, shows obvious variation, and observe most of strain system and take place obviously to separate.The whole obviously change short (Fig. 6 B) of No. 31, No. 39, No. 40, No. 41, No. 43 strains system; No. 38 strains system wherein 1 strain becomes short, and branch amount increases; No. 42 strains are most of plants stems principal deformation purple, partly crawl type, branch amount showed increased becomes close branch type, alternately bloom (Fig. 6 C); Other few part plant of homophyletic system are short and small, and No. 22 stem branches of mutagenesis parent Hua Yu are green, and plant type is upright; Dredge branch, bloom continuously (Fig. 6 A); Variation has also all taken place and has separated (Fig. 6 D) in other strain systems.
7. the drought resistance of resistance body is identified
M with 7 individual plants (No. 33, No. 34, No. 35, No. 37, No. 41, No. 42, No. 43) results
2The part seed in generation is seeded in plastic tunnel by strain system, compares for No. 20 with mutagenesis parent Hua Yu.After carrying out the arid processing seedling stage, most of resistant plant offspring grows normally; And flower is educated No. 20 growths and obviously suppressed, and is less, partial blade withered and yellow (Fig. 7).
The method that said arid is handled is: planting seed in plastic tunnel, is watered sufficient water during sowing, 1 first quarter moon does not water afterwards, carries out arid and handles (SOD, POD measure and draw materials is the blade material of getting this moment).
8. the mensuration of the physiological and biochemical index of resistance body
8.1 thick enzyme extraction
Get plastic tunnel and carry out the plant (M after arid is handled
2Generation) blade takes by weighing bright leaf sample 0.5g in the mortar of precooling, adds little P VP and silica, and the phosphate buffer (pH7.8) that adds 1ml 0.05mol/L grinds pulping on ice bath, and making final volume with buffer solution is 5ml.In 10000 rev/mins of refrigerated centrifuge 20min, 4 ℃ of preservations of supernatant are subsequent use with extract.
8.2 the mensuration that superoxide dismutase (SOD) is active
Measuring superoxide dismutase (SOD) activity with reference to the plant physiology experimental technique, is 1 enzyme unit to suppress 50% of NBT photochemical reduction.
SOD gross activity (U/g)=[(Ack-AE) * V]/(0.5Ack * W * Vt)
Ack is the absorbance of irradiation control tube; AE is the absorbance of sample cell; V is sample liquid cumulative volume (ml); W is the sample fresh weight; Amount of samples when Vt is mensuration.
The result is as shown in Figure 8, and as can beappreciated from fig. 8, the SOD activity of No. 33, No. 34, No. 35, No. 41, No. 42 5 strains system is higher than No. 20, mutagenesis parent Hua Yu in 7 strains of test system, and special 33,34, No. 35 SOD are active obviously to raise, be the parent 2-3 doubly.No. 37 strain systems and parent are basic identical, and No. 43 strains system is starkly lower than the parent.
8.3 the mensuration that peroxidase (POD) is active
Measure peroxidase (POD) activity with reference to Zhang Zhi's good method, the oxidized speed of wooden phenol that heals in the record 5min, changing 0.01 with per minute absorbance A 470 is 1 peroxidase activity unit (U), is calculated as follows the POD activity:
POD active (U/ (g.min))=(Δ A470 * Vt)/(W * Vs * 0.01 * t)
Δ A470 is the variation of reaction time internal absorbance; Vt is extract cumulative volume (ml); W is sample fresh weight (g); Vs takes enzyme liquid long-pending (ml) when being mensuration; T is reaction time (min).
The result is as shown in Figure 9, is found out by Fig. 9, and in 7 strain systems of test, the POD activity of No. 33, No. 34, No. 35, No. 41 4 strains system is active high than No. 20 POD of mutagenesis parent Hua Yu.Can also be explained that by Fig. 4 and Fig. 5 SOD is active more consistent with POD determination of activity result, the SOD activity is higher than in No. 33, No. 34, No. 35, No. 41, No. 42 of parent, only has No. 42 POD activity to be lower than contrast, and other 4 strains are that the POD activity all is higher than the parent.And No. 43 strains are that SOD activity and POD activity are all minimum in the material of test, all are starkly lower than the parent.
PYM is a kind of antibiotic, as a kind of new mutagen, be proved to be have safety, efficient, characteristics such as mutagenic frequency is high, scope is big, have wide development and application prospect.The present invention utilizes PYM as mutagen, and the HYP resistance seedling of acquisition is through the graft and transplantation field, and 13 strains obtain seed.Next year is seeded in the land for growing field crops by strain system, and 12 strain systems morph and separate.The result has further confirmed the mutagenesis effect of PYM.
POD can remove oxygen radical in the organism, avoids histocyte to come to harm, and coerces, plays a part in anti-ageing very important in the degeneration-resistant border of biology.POD is strong more at the vigor of rising proof cell in adverse circumstance of cell intensive amount.The major function of SOD also is to remove superoxide ion group in the organism, the injury of defence active oxygen or other peroxide radical cell membrane.Jiang Huifang etc. measure SOD under the peanut drought stress active, and the result shows that the active rising of the SOD of drought-resistant variety is more; And the active rising of the SOD of sensitive varieties is less.Mutagenic obtained HYP resistant plant exsomatize in this research behind the graft and transplantation field, the M of 7 individual plant results
2The part seed in generation, next year is seeded in plastic tunnel by strain system, carry out arid processing seedling stage after, most of resistant plant offspring grows normally; And No. 20 growths of mutagenesis parent Hua Yu are obviously suppressed.SOD is active to be shown with POD determination of activity result, most of strain be the active and POD activity of SOD apparently higher than the mutagenesis parent, explain that these resistance seedlings of acquisition are degeneration-resistant mutant.
Proline has important function as the osmotic adjustment material in salt tolerant, Physiology of Drought Resistance.,, successively on many crops, obtain the mutant system of anti-HYP, and show stronger resistance as selection pressure with proline analogs-hydroxyproline (HYP) through the tissue culture means.This research makes an addition to HYP in the peanut body embryo germination medium carries out directed screening, has finally obtained the drought resisting mutant, further specifies HYP and makes an addition to as screening pressure that the degeneration-resistant mutant of directed screening is effective in the medium.Directed screening can be saved human and material resources, financial resources.
The present invention utilizes safety, PYM carries out the stripped mutagenesis of peanut as mutagen efficiently, and as screening pressure, the resistance regrowth of acquisition is through the graft and transplantation field with HYP, and 13 plant have obtained mature seed.Next year is by strain system sowing, and wherein 12 strain systems morph and separate, and explain that sudden change has taken place gene; After 7 strain systems carry out the arid processing; Most of plant performance growth is normal, and the mutagenesis parent grows and obviously suppressed, and 5 strains are that the SOD activity is higher than the mutagenesis parent in 7 strains systems; Wherein 4 strains are that the POD activity also is higher than the mutagenesis parent, confirm that these mutant have stronger drought-resistant ability.The salt resistance of these mutant, cold resistance await further research.
Claims (8)
1. the method for mutagenesis directed screening resistance body of exsomatizing of cultivating peanut is characterized in that it comprises the steps:
(1) go the embryo behind the cotyledon to carry out surface sterilization ripe peanut seed;
(2) the embryo leaflet of the said embryo of separation is an explant under aseptic condition, and the embryo leaflet is inoculated in the body embryonal induction medium, carries out mutagenesis and cultivates 25～30d, few part explant organizator embryo; Said body embryonal induction medium is with MSB
5Be minimal medium, and add 3～4mg/L bleomycin A5 and 5～10mg/L 2,4-D;
(3) explant of organizator embryo is transferred on the body embryo germination medium cultivates, carry out directed degeneration-resistant screening and obtain the resistance regrowth, said body embryo germination medium is with MSB
5Be minimal medium, and add 4～8mg/L hydroxyproline and 4mg/L BAP;
(4) when the resistance regrowth grows to 2～3cm, downcut from base portion, graft on the plumular axis of peanut seedling of axenic germination, behind the aseptic culture 1-2d, transplant acclimation shaking culture in seedling medium, after be transplanted to the field.
2. the exsomatize method of the degeneration-resistant mutant of mutagenesis directed screening of peanut according to claim 1 is characterized in that: adopt in the said step (3) to add screening and press and do not add screening and press and respectively cultivate 4w and replace cultivation.
3. the method for the stripped degeneration-resistant mutant of mutagenesis directed screening of peanut according to claim 2; It is characterized in that: the degeneration-resistant screening concentration of hydroxyproline in body embryo germination stage is 4mg/L in the said step (3), and the degeneration-resistant screening concentration of hydroxyproline is 8mg/L during successive transfer culture.
4. the method for the stripped degeneration-resistant mutant of mutagenesis directed screening of peanut according to claim 1; It is characterized in that: alcohol-pickled 15～25s of 75% is adopted in the surface sterilization of peanut embryo in the said step (1); Soak with 0.1% mercuric chloride again; With after the rinsed with sterile water, place sterile water to soak 12～16h then.
5. the method for the stripped degeneration-resistant mutant of mutagenesis directed screening of peanut according to claim 1; It is characterized in that: condition of culture is in said step (2) and (3): temperature is that 24～26 ℃, intensity of illumination are that 2000～3000Lx, light application time are 10-15h/d, medium transfer to pH 5.8.
6. the method for the stripped degeneration-resistant mutant of mutagenesis directed screening of peanut according to claim 1, it is characterized in that: said body embryonal induction medium is MSB
5+ 5 mg/L 2,4-D+3mg/L PYM+30g/L sucrose+8g/L agar; Said body embryo germination medium is MSB
5+ 4 mg/L BAP+30g/L sucrose+8g/L agar+4mg/L hydroxyproline.
7. the method for the stripped degeneration-resistant mutant of mutagenesis directed screening of peanut according to claim 1, it is characterized in that: the peanut aseptic seedling with seedling age 9-12d in the said step (4) is a stock, the resistance regrowth is that aseptic grafting is carried out in scion.
8. the method for the stripped degeneration-resistant mutant of mutagenesis directed screening of peanut according to claim 1, it is characterized in that: the condition of said acclimation shaking culture is 22 ~ 26 ℃, 18～25d is cultivated in the domestication chamber.
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| CN2012102979385A Pending CN102792892A (en) | 2012-08-21 | 2012-08-21 | Method for directional screening of resistance body by peanut in-vitro mutation |
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| CN103070076A (en) * | 2013-02-05 | 2013-05-01 | 青岛农业大学 | Method for directionally screening salt-tolerant body through peanut in vitro mutagenesis |
| CN105103859A (en) * | 2015-08-17 | 2015-12-02 | 青岛农业大学 | Transplanting method for tissue-cultured peanut seedlings |
| CN105993936A (en) * | 2016-05-12 | 2016-10-12 | 青岛农业大学 | Method for in-vitro oriented screening and verification of acetochlor resistant bodies for peanuts |
| CN107667861A (en) * | 2017-10-23 | 2018-02-09 | 河南省农业科学院 | A kind of engrafting method of peanut tissue culture seedlings |
| CN108353790A (en) * | 2018-02-09 | 2018-08-03 | 青岛农业大学 | A kind of breeding method of peanut high-oil kind |
| CN111837957A (en) * | 2018-02-08 | 2020-10-30 | 青岛农业大学 | Culture medium for in-vitro directional screening of high-oil bodies of peanuts and screening method |
| CN113951139A (en) * | 2021-11-08 | 2022-01-21 | 四川农业大学 | Creation method of novel anti-adversity cymbidium floribundum |
| CN116941531A (en) * | 2023-08-24 | 2023-10-27 | 吉林省农业科学院 | Peanut tissue in-vitro mutagenesis grafting method |
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- 2012-08-21 CN CN2012102979385A patent/CN102792892A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103070076A (en) * | 2013-02-05 | 2013-05-01 | 青岛农业大学 | Method for directionally screening salt-tolerant body through peanut in vitro mutagenesis |
| CN103070076B (en) * | 2013-02-05 | 2016-05-04 | 青岛农业大学 | One method of cultivating peanut Vitro Mutation directed screening salt tolerant body |
| CN105103859A (en) * | 2015-08-17 | 2015-12-02 | 青岛农业大学 | Transplanting method for tissue-cultured peanut seedlings |
| CN105993936A (en) * | 2016-05-12 | 2016-10-12 | 青岛农业大学 | Method for in-vitro oriented screening and verification of acetochlor resistant bodies for peanuts |
| CN105993936B (en) * | 2016-05-12 | 2018-07-13 | 青岛农业大学 | One cultivate peanut in vitro directed screening and identify anti-acetochlor body method |
| CN107667861A (en) * | 2017-10-23 | 2018-02-09 | 河南省农业科学院 | A kind of engrafting method of peanut tissue culture seedlings |
| CN107667861B (en) * | 2017-10-23 | 2020-12-25 | 河南省农业科学院 | Grafting method of peanut tissue culture seedlings |
| CN111837957A (en) * | 2018-02-08 | 2020-10-30 | 青岛农业大学 | Culture medium for in-vitro directional screening of high-oil bodies of peanuts and screening method |
| CN108353790A (en) * | 2018-02-09 | 2018-08-03 | 青岛农业大学 | A kind of breeding method of peanut high-oil kind |
| CN113951139A (en) * | 2021-11-08 | 2022-01-21 | 四川农业大学 | Creation method of novel anti-adversity cymbidium floribundum |
| CN113951139B (en) * | 2021-11-08 | 2023-04-07 | 四川农业大学 | Creation method of novel anti-adversity cymbidium floribundum |
| CN116941531A (en) * | 2023-08-24 | 2023-10-27 | 吉林省农业科学院 | Peanut tissue in-vitro mutagenesis grafting method |
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