CN103444533B - Tissue culture method for increasing plant regeneration rate of wheat immature embryos - Google Patents

Abstract

The invention discloses a tissue culture method for increasing the regeneration rate of wheat immature embryos. The tissue culture method disclosed by the invention comprises the following steps: (1) putting the wheat immature embryos to be cultured on a callus induction culture medium for culturing, so as to obtain embryogenic calluses, wherein the callus induction culture medium contains hydrogen peroxide (H2O2) with a volume fraction of 0.00015-0.0003%; (2) transferring the embryogenic calluses to a differentiation culture medium for culturing, so as to obtain regenerated wheat plants. According to the method, the condition that the regeneration rate of the wheat immature embryos is increased obviously is found through adding H2O2 to the SD2 callus induction culture medium, and H2O2 is effective to wheat varieties, easy to regenerate, such as YM18, JD18 and CB031 and wheat varieties, difficult to regenerate, such as JM22, NC4 and YM20, so that the method has an important significance in improvement on good wheat varieties by means of genetic engineering and cell engineering.

Description

Improve the method for tissue culture of wheat immature embryo shoot regeneration frequency

Technical field

The invention belongs to field of plant tissue culture, relate to a kind of method for tissue culture improving wheat immature embryo regeneration rate.

Background technology

High Frequency of Plant Regeneraton is the basis of carrying out wheat cell Engineering Breeding and transgenic breeding.At present, the explant utilized in wheat cell Engineering Breeding and transgenic breeding is the tissue and organ that differentiation degree is lower substantially, as wheat immature embryo, mature embryo, young fringe, flower pesticide, microspore, apical meristem etc., also there is report with the Study on tissue culture that the organ that the differentiation degrees such as root, blade, coleoptile are higher is explant.The research data of current accumulation shows, wheat immature embryo is compared with other explants, and cultured in vitro callus induction rate and shoot regeneration frequency are all higher.In order to improve the efficiency of wheat immature embryo cultured in vitro regeneration plant further, Chinese scholars has studied the multiple factors affecting wheat immature embryo and cultivate in great detail, comprises genotype, plant hormone, rataria size, medium composition, carbon source, organic additive, oxygen concentration, dry process and cultivation program etc.Meanwhile, find the environmental condition of the wheat donor plant growths such as temperature, illumination, nutrition, humidity, affect its regeneration effect by the physiological status affecting wheat immature embryo.

Hydrogen peroxide (Hydrogen peroxide, H ₂o ₂) be the product of plant cell generation oxidation reaction, also be a kind of form producing active oxygen under plant suffers stress from outside situation, affect the normal metabolism of cell and grow, this impact has double action to plant cell: on the one hand, during generation oxidative stress, hydrogen peroxide is as a kind of signaling molecule, cause the synthesis of antioxidant reductase relevant in plant corpus, reduce the injury that adverse environmental factor causes; On the other hand, its cell membrane penetration of cell that oxidative stress occurs sexually revises, and then the disorder of trigger cell function, even causes cell death.The discoveries such as Tian, are formed in regeneration bud process with H at Callus Culture of Strawberry ₂o ₂produce, at early stage superoxide dismutase (the Superoxide dismutase of Calli Differentiation, SOD) increased activity, reduce gradually along with the dissociation of tissue, the first 5 days catalase (Catalase cultivated, CAT) sustained activity declines, and strengthens gradually subsequently, in the callus of low regeneration capacity, higher O detected ₂ ^-content and lower H ₂o ₂content, does not almost detect that SOD is active, shows H ₂o ₂take part in Callus Culture of Strawberry morphogenesis process, the effect of signaling molecule may be played in the former base forming process of bud.Find in matrimony vine blade tissue cultures, the H in Callus formation incipient cell ₂o ₂content increases, when adding 200 μMs of H in the medium ₂o ₂cultivate incidence rate of somatic embryo after 15 days and obtain maximum, the H of high concentration ₂o ₂(300 μMs) suppress the generation of embryo on the contrary, think H ₂o ₂affect the differentiation of callus, when there being appropriate H in cell ₂o ₂plant regeneration is conducive to, H when existing ₂o ₂may be take part in as signaling molecule the expression that matrimony vine embryo callus sends out related gene upper, stimulate the formation of cells,primordial.Find in the hypocotylar incubation of ice plant cotyledon, the H of low CAT activity and high concentration ₂o ₂be conducive to the differentiation of regeneration bud, H ₂o ₂the increase of concentration destroys original oxidation balance, thinks H ₂o ₂the expression with form generation related gene may be facilitated.These results suggest that, H ₂o ₂in plant embryos Callus formation and plant regeneration process, there is important function.But, about H ₂o ₂effect in wheat children cultivates and the research of suitable concentration thereof are not also reported.

Summary of the invention

The object of this invention is to provide a kind of method for tissue culture improving wheat immature embryo regeneration rate.

The method for tissue culture of raising wheat immature embryo regeneration rate provided by the present invention, specifically can comprise the steps:

(1) wheat immature embryo to be cultivated is placed on callus inducing medium cultivates, obtain embryo callus; Be the hydrogen peroxide (H of 0.0015-0.003 ‰ containing final volume mark in described callus inducing medium ₂o ₂);

(2) described embryo callus is transferred on differential medium cultivate, obtain regenerating wheat plant.

In the process, described wheat immature embryo can be heart-shaped phase rataria, and be specially the rear 13-14 days of pollination of blooming, diameter is the wheat immature embryo of 1.0-1.2mm.

In the present invention, described callus inducing medium is specially that in SD2 medium, add final volume mark be the medium obtained after the hydrogen peroxide of 0.0015-0.003 ‰.

In the process, the described hydrogen peroxide in described callus inducing medium adds when described SD2 medium is cooled to about 65 DEG C.

Wherein, described SD2 medium consists of: MS macroelement, MS trace element, MS molysite, VB ₁1.0mg/L, glutamine 150mg/L, 2,4-D2.0mg/L, sucrose 30g/L, plant gel (Phytagel) 2.4g/L, pH value 6.0.Each concentration is the final concentration of respective substance in described SD2 medium above.

Specifically, the solvent of described SD2 medium is water, solute and concentration as follows: NH ₄nO ₃1650mg/L, KNO ₃1900mg/L, CaCl ₂2H ₂o440mg/L, MgSO ₄7H ₂o370mg/L, KH ₂pO ₄1700mg/L, KI0.83mg/L, H ₃bO ₃6.2mg/L, MnSO ₄4H ₂o22.3mg/L, ZnSO ₄7H ₂o8.6mg/L, Na ₂moO ₄2H ₂o0.25mg/L, CuSO ₄5H ₂o0.025mg/L, CoCl ₂6H ₂o0.025mg/L, FeSO ₄7H ₂o27.8mg/L, Na ₂-EDTA2H ₂o37.3mg/L, sucrose 30g/L, Cobastab ₁1mg/L, glutamine 150mg/L, 2,4-D2.0mg/L, plant gel (Phytagel) 2.4g/L, pH value is 6.0.

In the process, described differential medium can be FHCK medium.

Wherein, consisting of of described FHCK medium: MS macroelement, MS trace element, MS molysite, MS vitamin, sucrose 20g/L, plant gel (Phytagel) 2.4g/L, pH value 6.0.Each concentration is the final concentration of respective substance in described FHCK medium above.

Specifically, the solvent of described FHCK medium is water, solute and concentration as follows: NH ₄nO ₃1650mg/L, KNO ₃1900mg/L, CaCl ₂2H ₂o440mg/L, MgSO ₄7H ₂o370mg/L, KH ₂pO ₄1700mg/L, KI0.83mg/L, H ₃bO ₃6.2mg/L, MnSO ₄4H ₂o22.3mg/L, ZnSO ₄7H ₂o8.6mg/L, Na ₂moO ₄2H ₂o0.25mg/L, CuSO ₄5H ₂o0.025mg/L, CoCl ₂6H ₂o0.025mg/L, FeSO ₄7H ₂o27.8mg/L, Na ₂-EDTA2H ₂o37.3mg/L, Cobastab ₁1mg/L, V _b60.5mg/L, nicotinic acid 0.5mg/L, glycine 2mg/L, inositol 100mg/L, sucrose 20g/L, plant gel (Phytagel) 2.4g/L, pH value is 6.0.

In the step (1) of described method, wheat immature embryo described to be cultivated is placed in (scultellum upward) on described callus inducing medium to cultivate, its condition of culture specifically can be: dark, temperature 24-26 DEG C (as 25 DEG C), time 20-25 days (as 21 days).

In the step (2) of described method, transferred to by described embryo callus on described differential medium and cultivate, its condition of culture specifically can be: illumination 10 hours/day, light intensity 3500Lx, temperature 24-26 DEG C (as 25 DEG C), time 15-25 days (as 21 days).

In the process, the kind of described wheat specifically can be at least one as follows: raise wheat 18, China spring, Jimai 22, capital winter 18, river agriculture 16, Ningchun4, raise wheat 20 and CB031.

More concrete, when described wheat for raise wheat 18, river agriculture 16, Ningchun4 or CB031 time, the final volume mark of hydrogen peroxide described in described callus inducing medium is with 0.0015-0.003 ‰ as well; When described wheat be China spring, Jimai 22 or when raising wheat 20, the final volume mark of hydrogen peroxide described in described callus inducing medium with 0.0015 ‰ as well.

Another object of the present invention is to provide a kind of medium.

Medium provided by the present invention is that in SD2 medium, add final volume mark be the medium obtained after the hydrogen peroxide of 0.0015-0.003 ‰.

Described medium can be used for improving described wheat immature embryo tissue culture plants regeneration rate.

In the present invention, the described hydrogen peroxide (H adopted ₂o ₂) stoste be Xilong Chemical Co., Ltd's product (AR level, CAS number: 7722-84-1), be the aqueous hydrogen peroxide solution of volume fraction 30%.

All above described " in described SD2 medium, adding the hydrogen peroxide that final volume mark is 0.0015-0.003 ‰ ", be: in described SD2 medium, add aqueous hydrogen peroxide solution (100 times of dilution gained being carried out to the aqueous hydrogen peroxide solution of volume fraction 30% with the water) (H that volume fraction is 0.3% ₂o ₂stock solution), make the final volume mark of described hydrogen peroxide be 0.0015-0.003 ‰.

In callus inducing medium, H is not added in existing wheat immature embryo cultural method ₂o ₂step, the present invention cultivates in callus inducing medium the H adding different volumes percentage at wheat immature embryo ₂o ₂, at suitable H ₂o ₂significantly improve multiple wheat genotypes IMMATURE EMBRYOS CULTURE shoot regeneration frequency under consumption, there is good actual application value.

Experiment proves, utilizes the inventive method to raise wheat 18 rataria at the H adding different volumes percentage to wheat breed ₂o ₂the SD2 callus inducing medium of (0,0.0015 ‰, 0.003 ‰ and 0.0045 ‰) is cultivated, research H ₂o ₂on the impact of wheat immature embryo regeneration.Found that, work as H ₂o ₂when volume fraction is 0.0015 ‰, 0.003 ‰, regeneration rate is respectively 365.56%, 419.05%, and does not add H ₂o ₂contrast (regeneration rate is 141.18%) compare and be significantly increased, H ₂o ₂when volume fraction is 0.0045 ‰, shoot regeneration frequency is lower than contrast, shows H in wheat immature embryo callus inducing medium ₂o ₂optimum be 0.0015-0.003 ‰.The present inventor utilizes the method to wheat breed China spring, Jimai 22, capital winter 18, river agriculture 16, Ningchun4 further, raises wheat 20 and CB031 rataria at interpolation Optimum H ₂o ₂the improvement SD2 medium of (volume fraction 0.0015 ‰ and 0.003 ‰) is cultivated, not add H ₂o ₂sD2 medium be contrast, found that, add the H of volume fraction 0.0015 ‰ ₂o ₂significantly improve wheat breed China spring, Jimai 22, river agriculture 16, Ningchun4, raise wheat 20 and CB031 IMMATURE EMBRYOS CULTURE shoot regeneration frequency, add the H of volume fraction 0.003 ‰ ₂o ₂significantly improve wheat breed river agriculture 16, Ningchun4, CB031 IMMATURE EMBRYOS CULTURE shoot regeneration frequency.Show in wheat immature embryo callus inducing medium, to add suitable concentration Optimum H ₂o ₂(volume fraction 0.0015 ‰ and 0.003 ‰) can improve most wheat breed IMMATURE EMBRYOS CULTURE shoot regeneration frequency, and have prevalent effects, Wheat Cultivars is suitable for the H added ₂o ₂concentration is slightly different.

The present invention by adding H in SD2 callus inducing medium ₂o ₂find that wheat immature embryo regeneration rate significantly improves, to regeneration be easy to wheat breed as raise the more difficult wheat breed of wheat 18, capital winter 18, CB031 and regeneration as Jimai 22, Ningchun4, to raise wheat 20 all effective, this is significant for utilizing gene engineering approach and cell engineering approach to improve excellent wheat breed.

Accompanying drawing explanation

Fig. 1 is different amounts H ₂o ₂wheat breed is raised to the impact of wheat 18 rataria regeneration effect.Wherein, A represents containing volume fraction 0 ‰ H ₂o ₂medium on regeneration effect; B represents containing volume fraction 0.0015 ‰ H ₂o ₂medium on regeneration effect; C represents containing volume fraction 0.003 ‰ H ₂o ₂medium on regeneration effect; D represents containing volume fraction 0.0045 ‰ H ₂o ₂medium on regeneration effect.

Fig. 2 is that Wheat Cultivars is at suitable concentration H ₂o ₂under regeneration effect compare.Wherein, A represents that China spring is at control medium (volume fraction 0 ‰ H ₂o ₂) on regeneration effect; B represents that China spring is containing volume fraction 0.0015 ‰ H ₂o ₂medium on regeneration effect; C represents that Jimai 22 is at control medium (volume fraction 0 ‰ H ₂o ₂) on regeneration effect; D represents that Jimai 22 is containing volume fraction 0.0015 ‰ H ₂o ₂medium on regeneration effect; E represents that river agriculture 16 is at control medium (volume fraction 0 ‰ H ₂o ₂) on regeneration effect; F represents that river agriculture 16 is containing volume fraction 0.0015 ‰ H ₂o ₂medium on regeneration effect; G represents that Ningchun4 is at control medium (volume fraction 0 ‰ H ₂o ₂) on regeneration effect; H represents that Ningchun4 is containing volume fraction 0.0015 ‰ H ₂o ₂medium on regeneration effect; I represents and raises wheat 20 at control medium (volume fraction 0 ‰ H ₂o ₂) on regeneration effect; J represents that raising wheat 20 is containing volume fraction 0.0015 ‰ H ₂o ₂medium on regeneration effect.Q represents that CB031 is at control medium (volume fraction 0 ‰ H ₂o ₂) on regeneration effect; L represents that CB031 is containing volume fraction 0.0015 ‰ H ₂o ₂medium on regeneration effect.

Embodiment

The experimental technique used in following embodiment if no special instructions, is conventional method.

Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.

The definition of wheat immature embryo: the wheat immature embryo after the pollination of wheat ovary, the tender seed of the growth wheat of 13-14 days children stripped.

Wheat breed is raised wheat 18, China spring, Jimai 22, capital winter 18, river agriculture 16, Ningchun4, is raised wheat 20 and CB031: Institute of Crop Science, Chinese Academy of Agricultural Science's crop germplasm resource protection gratuitously provides to society with research center.

Wheat breed China spring, Ningchun4: be recorded in " Shi Zhenyuan etc. the above the average age for marriage rataria regenerability of wheat is improved and Agrobacterium-mediated Transformation. Scientia Agricultura Sinica, 2011,44 (2): 225-232 " in a literary composition;

Wheat breed raises wheat 18: be recorded in " Li Xin etc. 12 new variety of wheat mature embryo regeneration performances and Agrobacterium infect sensitivity assessment. Chinese agriculture science and technology Leader, 2012,14 (2): 40-46 " in a literary composition;

Wheat breed Jimai 22: be recorded in " Tao et al.Improvement of Plant Regeneration from Immature Embryos of Wheat Infected Agrobacterium tumefaciens.Agricultural Sciences in China; 2011,10 (3): 317-326 " literary composition;

The wheat breed capital winter 18: be recorded in " single Fu Hua etc. state examines the seed selection in New Winter Wheat Variety capital winter 18. Beijing Agriculture, 2012,6:20-21 " in a literary composition;

Wheat breed river agriculture 16: be recorded in " Li Wei etc. the Molecular Identification of New Wheat Variety Chuannong 16. wheat crops journal, 2004,24 (1): 6-10 " in a literary composition;

Wheat breed raises wheat 20: be recorded in " Ding Jinfeng etc. later stage nitrogen applying stage spends the impact of rear Biomass production power and output on raising wheat 20. Yangzhou University's journal (agricultural with life science version), 2012,33 (3): 56-62 " in a literary composition;

Wheat breed CB031: be recorded in " Zeng Xiangyan etc. the improvement of wheat Pyramiding line YW243 and utilization. Acta Agronomica Sinica, the 05th phase in 2006 " in a literary composition;

Wherein, wheat breed China spring, Jimai 22 and Ningchun4 belong to the lower wheat genotypes of rataria regeneration power, refer to bibliography " She et al.Efficient Regeneration Porential is Closely Related to Auxin Exposure Time and Catalase Metabolism During the Somatic Embryogenesis of Immature Embryos in Triticum aestivum L.Molecular Biotechnology; 2013,54:451-460 ".

SD2 medium: solvent is water, solute and concentration as follows: NH ₄nO ₃1650mg/L, KNO ₃1900mg/L, CaCl ₂2H ₂o440mg/L, MgSO ₄7H ₂o370mg/L, KH ₂pO ₄1700mg/L, KI0.83mg/L, H ₃bO ₃6.2mg/L, MnSO ₄4H ₂o22.3mg/L, ZnSO ₄7H ₂o8.6mg/L, Na ₂moO ₄2H ₂o0.25mg/L, CuSO ₄5H ₂o0.025mg/L, CoCl ₂6H ₂o0.025mg/L, FeSO ₄7H ₂o27.8mg/L, Na ₂-EDTA2H ₂o37.3mg/L, sucrose 30g/L, Cobastab ₁1mg/L, glutamine 150mg/L, 2,4-D2.0mg/L, plant gel (Phytagel) 2.4g/L, pH value is 6.0.Adopt 121 DEG C of high pressure moist heat sterilizations 15 minutes.

FHCK medium: solvent is water, solute and concentration as follows: NH ₄nO ₃1650mg/L, KNO ₃1900mg/L, CaCl ₂2H ₂o440mg/L, MgSO ₄7H ₂o370mg/L, KH ₂pO ₄1700mg/L, KI0.83mg/L, H ₃bO ₃6.2mg/L, MnSO ₄4H ₂o22.3mg/L, ZnSO ₄7H ₂o8.6mg/L, Na ₂moO ₄2H ₂o0.25mg/L, CuSO ₄5H ₂o0.025mg/L, CoCl ₂6H ₂o0.025mg/L, FeSO ₄7H ₂o27.8mg/L, Na ₂-EDTA2H ₂o37.3mg/L, Cobastab ₁1mg/L, V _b60.5mg/L, nicotinic acid 0.5mg/L, glycine 2mg/L, inositol 100mg/L, sucrose 20g/L, plant gel (Phytagel) 2.4g/L, pH value is 6.0.Adopt 121 DEG C of high pressure moist heat sterilizations 15 minutes.

Hydrogen peroxide stoste: (AR level, CAS numbers: 7722-84-1), be the aqueous hydrogen peroxide solution of volume fraction 30% for Xilong Chemical Co., Ltd's product.200 μ l H are accurately drawn with micropipettor ₂o ₂stoste, joins in 20ml distilled water, and it is the stock solution of 0.3% that preparation obtains hydrogen peroxide volume percentage, with the filtering with microporous membrane sterilizing of 0.25 μm of specification, keeps in Dark Place in 4 DEG C of refrigerators.During use, after SD2 medium high pressure moist heat sterilization, be at room temperature cooled to about 65 DEG C, add the H of different amounts sterilizing after filtration according to process ₂o ₂stock solution.

Different amounts H is added in embodiment 1, SD2 medium ₂o ₂wheat is raised to the impact of wheat 18 rataria regeneration effect

In the present embodiment, raise wheat 18 for subjects with wheat breed, the tissue culture procedures of its wheat immature embryo is as follows:

One, the acquisition of wheat children tassel

Wheat is raised wheat 18 and be planted in Institute of Crop Science, Chinese Academy of Agricultural Science greenhouse (in September, 2011), bloom 13-14 days (in January, 2012) after pollinating, collect wheat and raise the wheat 18 prematurity wheat head, now rataria is heart-shaped phase rataria, and its diameter is 1.0-1.2mm.

Two, wheat prematurity seed sterilizing

Raise the wheat 18 prematurity wheat head from wheat and get its prematurity seed, 70%(volume fraction) alcohol surface sterilization 1 minute, 15%(volume fraction) clorox sterilizing 15-20 minute, aseptic water washing 4-5 time.

Three, tissue cultures

1, Fiber differentiation produces embryo callus

On prematurity seed, cut embryo point with sterilized knife blade, then taken out by wheat immature embryo, scultellum is seeded to upward and adds different final volume mark H ₂o ₂on the SD2 medium of (0,0.0015 ‰, 0.003 ‰ and 0.0045 ‰), each culture dish inoculation 30-35 piece of wheat immature embryo, not add H ₂o ₂sD2 medium be contrast, 25 DEG C, cultivate 21 days induced embryonic callus under dark condition.

2, break up cultivation and obtain Regenerated Plantlets of Wheat

Embryo callus step 1 obtained is transferred on FHCK medium and is cultivated 21 days (light application time is 10 hours every days, and intensity of illumination is 3500Lx, and temperature is 25 DEG C), and differentiation obtains wheat regenerated green bud.

Therebetween, record inoculation rataria number, statistics callus number, embryo callus number, differentiation callus number, regeneration bud number, frequency of embryonic callus induction, differentiation callus rate, regeneration rate.Experiment establishes 3 repetitions, results averaged.

Frequency of embryonic callus induction (%)=(embryo callus number ÷ inoculates rataria number) × 100

Differentiation callus rate (%)=(differentiation callus number ÷ inoculates rataria number) × 100

Regeneration rate (%)=(regeneration bud number ÷ inoculates rataria number) × 100

Four, variable concentrations H ₂o ₂to the comparison of wheat immature embryo regeneration effect

Wheat raises wheat 18 rataria through containing variable concentrations H ₂o ₂regenerated outcome after medium is cultivated is as shown in table 1 and Fig. 1.As can be seen from the table, as H in medium ₂o ₂when volume fraction is 0.0015 ‰, 0.003 ‰, regeneration rate is respectively 365.56%, 419.05%, does not add H ₂o ₂contrast regeneration rate be that 141.18%, 2 process compare according to being significantly increased, difference reaches significance level (P<0.05).But as H in medium ₂o ₂when volume fraction is 0.0045 ‰, shoot regeneration frequency is 121.9%, a little less than contrast, does not have significant difference.Show H in wheat immature embryo callus inducing medium ₂o ₂optimum be 0.0015-0.003 ‰.

Table 1 different amounts H ₂o ₂wheat breed is raised to the impact of wheat 18 IMMATURE EMBRYOS CULTURE regeneration effect

Note: different lowercase alphabets shows significant difference (P<0.05).

Embodiment 2, suitable concentration H ₂o ₂on the impact of Wheat Cultivars rataria regeneration effect

In the present embodiment, with wheat breed China spring, Jimai 22, capital winter 18, river agriculture 16, Ningchun4, raise wheat 20 and CB031 for subjects, the tissue culture procedures of its wheat immature embryo is as follows:

One, the acquisition of wheat children tassel

By wheat China spring, Jimai 22, capital winter 18, river agriculture 16, Ningchun4, raise wheat 20 and CB031 is planted in Institute of Crop Science, Chinese Academy of Agricultural Science's experimental plot wheat experimental field (in October, 2011), bloom 13-14 days (in May, 2012) after pollinating, collect wheat China spring, Jimai 22, capital winter 18, river agriculture 16, Ningchun4, raise wheat 20 and the CB031 prematurity wheat head, now rataria is heart-shaped phase rataria, and its diameter is 1.0-1.2mm.

Two, wheat prematurity seed sterilizing

From wheat China spring, Jimai 22, capital winter 18, river agriculture 16, Ningchun4, raise wheat 20 and the CB031 prematurity wheat head gets its prematurity seed, 70%(volume fraction) alcohol surface sterilization 1 minute, 15%(volume fraction) clorox sterilizing 15-20 minute, aseptic water washing 4-5 time.

Three, tissue cultures

1, Fiber differentiation produces embryo callus

With sterilized knife blade at China spring, Jimai 22, capital winter 18, river agriculture 16, Ningchun4, raise on wheat 20 and CB031 prematurity seed and cut embryo point, then taken out by wheat immature embryo, scultellum is seeded to upward and adds Optimum H ₂o ₂on the SD2 callus inducing medium of (volume fraction 0.0015 ‰ or 0.003 ‰), each culture dish inoculation 30-35 piece of wheat immature embryo, not add H ₂o ₂sD2 medium be contrast, 25 DEG C, cultivate 21 days induced embryonic callus under dark condition.

2, break up cultivation and obtain Regenerated Plantlets of Wheat

Transferred to by step 1 gained embryo callus on FHCK medium and cultivate 21 days (light application time is 10 hours every days, and intensity of illumination is 3500Lx, and temperature is 25 DEG C), differentiation obtains wheat regenerated green bud.

Therebetween, record inoculation rataria number, statistics callus number, embryo callus number, differentiation callus number, regeneration bud number, regeneration rate.Experiment establishes 3 repetitions, results averaged.

Regeneration rate (%)=(regeneration bud number ÷ inoculates rataria number) × 100

Four, Optimum H ₂o ₂to the comparison of Wheat Cultivars rataria regeneration effect

7 wheat breed China spring, Jimai 22, capital winter 18, river agriculture 16, Ningchun4s, raise wheat 20 and CB031 rataria at interpolation Optimum H ₂o ₂sD2 callus inducing medium on cultivate after regenerated outcome as shown in table 2 and Fig. 2.As can be seen from the table, the H of volume fraction 0.0015-0.003 ‰ is added in wheat immature embryo callus inducing medium ₂o ₂have raising wheat immature embryo regeneration rate and generally act on, but Wheat Cultivars is suitable for the H that adds ₂o ₂amount slightly different, add volume fraction 0.0015 ‰ H in callus inducing medium ₂o ₂significantly improve wheat China spring, Jimai 22, river agriculture 16, Ningchun4, raise the regeneration rate of wheat 20 and CB031 rataria, in callus inducing medium, add the H of volume fraction 0.003 ‰ ₂o ₂significantly improve the regeneration rate of wheat river agriculture 16, Ningchun4 and CB031 rataria.

Table 2 different amounts H ₂o ₂on the impact of Wheat Cultivars IMMATURE EMBRYOS CULTURE regeneration effect

Note: different lowercase alphabets shows significant difference (P<0.05).

Claims (6)

1. improve a method for tissue culture for wheat immature embryo regeneration rate, comprise the steps:

(1) wheat immature embryo to be cultivated is placed on callus inducing medium cultivates, obtain embryo callus;

Described callus inducing medium is that in SD2 medium, add final volume mark be the medium obtained after the hydrogen peroxide of 0.0015-0.003 ‰;

The solvent of described SD2 medium is water, solute and concentration as follows: NH ₄nO ₃1650mg/L, KNO ₃1900mg/L, CaCl ₂2H ₂o 440mg/L, MgSO ₄7H ₂o 370mg/L, KH ₂pO ₄1700mg/L, KI 0.83mg/L, H ₃bO ₃6.2mg/L, MnSO ₄4H ₂o 22.3mg/L, ZnSO ₄7H ₂o 8.6mg/L, Na ₂moO ₄2H ₂o 0.25mg/L, CuSO ₄5H ₂o 0.025mg/L, CoCl ₂6H ₂o 0.025mg/L, FeSO ₄7H ₂o 27.8mg/L, Na ₂-EDTA2H ₂o 37.3mg/L, sucrose 30g/L, Cobastab ₁1mg/L, glutamine 150mg/L, 2,4-D2.0mg/L, plant gel 2.4g/L, pH value is 6.0;

(2) described embryo callus is transferred on differential medium cultivate, obtain regenerating wheat plant;

Described differential medium is FHCK medium;

The solvent of described FHCK medium is water, solute and concentration as follows: NH ₄nO ₃1650mg/L, KNO ₃1900mg/L, CaCl ₂2H ₂o 440mg/L, MgSO ₄7H ₂o 370mg/L, KH ₂pO ₄1700mg/L, KI 0.83mg/L, H ₃bO ₃6.2mg/L, MnSO ₄4H ₂o 22.3mg/L, ZnSO ₄7H ₂o 8.6mg/L, Na ₂moO ₄2H ₂o0.25mg/L, CuSO ₄5H ₂o 0.025mg/L, CoCl ₂6H ₂o 0.025mg/L, FeSO ₄7H ₂o 27.8mg/L, Na ₂-EDTA2H ₂o 37.3mg/L, Cobastab ₁1mg/L, V _b60.5mg/L, nicotinic acid 0.5mg/L, glycine 2mg/L, inositol 100mg/L, sucrose 20g/L, plant gel 2.4g/L, pH value is 6.0;

The kind of described wheat be following in any one: raise wheat 18, China spring, Jimai 22, river agriculture 16, Ningchun4, raise wheat 20 and CB031.

2. method according to claim 1, is characterized in that: described wheat immature embryo is heart-shaped phase rataria.

3. method according to claim 1, it is characterized in that: in step (1), be placed in by wheat immature embryo described to be cultivated on described callus inducing medium and cultivate, condition of culture is: dark, temperature 24-26 DEG C, time 20-25 days.

4. method according to claim 1, it is characterized in that: in step (2), transferred to by described embryo callus on described differential medium and cultivate, condition of culture is: illumination 10 hours/day, light intensity 3500Lx, temperature 24-26 DEG C, time 15-25 days.

5. for improving the medium of wheat immature embryo regeneration rate, it is characterized in that: described medium is that in SD2 medium, add final volume mark be the medium obtained after the hydrogen peroxide of 0.0015-0.003 ‰;

The solvent of described SD2 medium is water, solute and concentration as follows: NH ₄nO ₃1650mg/L, KNO ₃1900mg/L, CaCl ₂2H ₂o 440mg/L, MgSO ₄7H ₂o 370mg/L, KH ₂pO ₄1700mg/L, KI 0.83mg/L, H ₃bO ₃6.2mg/L, MnSO ₄4H ₂o 22.3mg/L, ZnSO ₄7H ₂o 8.6mg/L, Na ₂moO ₄2H ₂o 0.25mg/L, CuSO ₄5H ₂o 0.025mg/L, CoCl ₂6H ₂o 0.025mg/L, FeSO ₄7H ₂o 27.8mg/L, Na ₂-EDTA2H ₂o 37.3mg/L, sucrose 30g/L, Cobastab ₁1mg/L, glutamine 150mg/L, 2,4-D2.0mg/L, plant gel 2.4g/L, pH value is 6.0;

The kind of described wheat be following in any one: raise wheat 18, China spring, Jimai 22, river agriculture 16, Ningchun4, raise wheat 20 and CB031.

6. medium described in claim 5 is improving the application in wheat immature embryo tissue culture plants regeneration rate.