CN100559933C - A method for obtaining a large amount of tall fescue regenerated plants through tissue culture - Google Patents

Abstract

The invention proposes a method for obtaining a large amount of tall fescue regenerated plants through tissue culture. The method comprises the steps of using mature tall fescue seeds as explants, forming callus tissue on an improved induction medium after disinfection, and then inducing the occurrence of somatic embryos, and then taking the callus with somatic embryos The tissue is inoculated on the improved differentiation medium to induce the germination of the somatic embryo and the callus to continue to differentiate to form a complete regenerated plant with roots and shoots, and finally transplanted to the flower pot to complete the whole process of tall fescue plant regeneration. The method has general adaptability to regeneration of most tall fescue varieties, high frequency of obtaining tall fescue regenerated plants, short culture period, and is suitable for genetic improvement of tall fescue.

Description

A kind of method that obtains a large amount of Festuca Arundinacea regeneration plants by tissue culture

Technical field

The present invention relates to utilize tissue culture technique to obtain the method for a large amount of Festuca Arundinacea regrowths, belong to biological technical field.

Background technology

Festuca Arundinacea is a kind of in the world important high nutrition cold season forage grass, also is a kind of important turfgrass.It has the characteristics of drought resisting, barren-resistant, disease-resistant and wide adaptability, extensive use in the planting of urban afforestation and sports turf.But it also have blade coarse, do not have stolon, the lawn autgmentability is poor, summer shortcoming such as poor growth.Utilize biotechnology can be efficiently, apace the crop varieties proterties is carried out genetic improvement, thereby provide a kind of convenient, fast and effective method for the Festuca Arundinacea breeding.Because the key of biotechnology breeding is to set up a tissue culturing system efficiently, so that the culture systems of recipient cell behind the genetic recipient system that intends the improvement objective trait and the genetic transformation to be provided.

The research of Festuca Arundinacea tissue culture aspect starts from the eighties in 20th century.1979, Lowe and Conger (Crop Sci, 1979,19,397～400) were from Festuca Arundinacea mature seed embryo evoked callus and obtain regeneration plant successfully.After this, people cultivate (Lowe etc., Crop Sci by pollen sac, 1979,19,397～400), young fringe is cultivated (Dale etc., Z P flanzen physiol Bd, 1983,111:39～45), obtain the cultivation (Dalton etc. of protoplast from suspension cell, Plant Physiol, 1988,132,170～175) and the mature seed evoked callus cultivate (Zhi Yuee etc., Shanghai Agricultural College's journal, 1998,16 (1), 46～48) etc. all obtained regeneration plant.(Chinese grassland, 2001,24 (1), 46～49) such as domestic money Haifeng county also reported Festuca Arundinacea tissue culture aspect, and (number/callus of sprouting inoculation piece number is up to 81.8% but its regeneration rate is lower; The method of this calculating differentiation rule is wrong).In addition, abroad the regeneration frequency of the Testuca arundinacea regenerating system plant of all reports is all lower, and the highest only is 71.6% (callus of sprouting piece number/inoculation callus piece number, Plant Breeding such as Bai, 2001,120,239～242).Therefore, for carrying out the efficient genetic improvement of Festuca Arundinacea, the high-frequency plant regenerating system research of setting up Festuca Arundinacea is necessary.

The present invention is directed to disadvantages of background technology and defective, overcome regeneration difficulty in the Festuca Arundinacea tissue culture procedures, and problem such as regeneration capacity significant difference between kind, by improveing the medium (MS that has reported, SH) component, design the medium that is fit to the most of kind regeneration of Festuca Arundinacea, and can control the callus state of Festuca Arundinacea and grow direction by certain experiment flow, increased substantially the pick-up rate (every callus can obtain more than 30 regrowth on the differential medium) of Festuca Arundinacea differentiation of calli rate (can reach 96%) and regeneration plant, and the time of finishing Testuca arundinacea regeneration only is about 10 weeks.For Festuca Arundinacea breed improvement and directed biotechnology breeding have been established solid foundation.Have certain social benefit and economic benefit, can promote the grassland industry development of China.

Summary of the invention

The present invention proposes a kind ofly to obtain the method for a large amount of Festuca Arundinacea regeneration plants by tissue culture, and the main contents of invention have: be suitable for Festuca Arundinacea in the minimal medium of giving birth to, medium proportioning, somatic embryo inducement and the differentiation of inducing Yu keeping of callus.

(1) minimal medium of the Festuca Arundinacea tissue culture minimal medium that is used for the Festuca Arundinacea tissue culture comprises modified MS medium (MSM) and improvement SH medium (MSH).The basis of MSM medium is MS medium (Murashige﹠amp; Skoog medium, 1962, Physiol.Plant 15:473-497) mineral salt, additional 2mg/L caseinhydrolysate, 9.9mg/L VB ₁, 9.5mg/L VB ₆, 4.5mg/L niacin (VB ₃), 8g/L agar and 30g/L sucrose.The composition of MSH medium is SH medium (Sckenk﹠amp; Hilderebrantdt, 1972, Can J Bot, the molysite of macroelement 50:199-204)+MS medium and trace element+caseinhydrolysate 1-2mg/L+VB ₁9.9mg/L+VB ₆9.5mg/L+ niacin (VB ₃) 4.5mg/L+ agar 8g/L+1%-2% sucrose.

(2) seed of inducing and keeping medium to disinfect of Festuca Arundinacea callus is inoculated into the additional 5～10mg/L 2 of MSM, and (kinetin carries out callus of induce on callus of induce medium KT) for 4-D and 0.025mg/L kinetin.Callus is transferred to subculture medium MSM+4.5mg/L 2 with it after forming for two weeks, carries out enrichment culture on 4-D+0.1～0.2mg/L KT.

(3) inducing with callus of somatic embryo broken up before carrying out the callus differentiation, carried out somatic embryo inducement earlier.The composition of Festuca Arundinacea callus somatic induction medium is MSM+2mg/L 2,4-D+0.1～0.2mg/L KT (concentration of sucrose is 3%).It is very fast that somatic embryo forms speed on this medium, and behind the cultivation 20d, the somatic embryo inducement rate generally reaches more than 60%.Festuca Arundinacea callus differentiation culture based component is MSH+2mg/L KT+1%～2% sucrose.The callus that will have embryoid is transferred to differential medium and was differentiated to form with regard to having a large amount of regeneration buds about last 20 day.

The present invention has compared obvious improvement with the document of having reported about the Festuca Arundinacea tissue culture.At first, (MSM is a reported first MSH) to the improved culture medium of mentioning among the present invention, and the medium of proof after improvement is more suitable for Festuca Arundinacea regeneration in experiment.In addition, the culture technique flow process that the present invention relates to and the Festuca Arundinacea regeneration effect of report relatively have significant superiority, because, under techniqueflow of the present invention, Festuca Arundinacea only needs about 10 weeks from the seed explant to regrowth, regeneration rate can reach 96%, can form more than 30 regeneration bud on the every callus, is a regenerating system that is fit to the Festuca Arundinacea genetic improvement.

The method implementation step that obtains a large amount of regeneration plants about the method for Festuca Arundinacea tissue culture is as follows:

(1) selection of explant: choosing full Festuca Arundinacea (Festucaarundinacea) seed in the experiment of the present invention is explant.

(2) inducing and keep of callus: choose full tall fescue seed, remove shell with sand papering, in 75% alcohol, soak 3min after, the 10min that sterilizes of the mercury chloride with 0.1% then, thoroughly washes 5 times with sterile distilled water.(MSM+5～10mg/L 2 to be inoculated into the callus of induce medium, 4-D and 0.025mg/L KT) on, two all callus form, and the callus that forms is transferred to callus maintenance medium, and (MSM+4.5mg/L 2,4-D+0.1 propagation (Fig. 1,2)～0.2mg/L KT+3% sucrose).

(3) the inducing and break up of somatic embryo: the callus enrichment culture (at this moment, reached the soybean grain size) after 20 days, will carry out somatic embryo inducement (Fig. 3) for improving regeneration rate.Callus is divided into about the 3mm size, be transferred to and carry out somatic embryo inducement on the somatic embryo inducement medium.After about 20 days (at this moment, the somatic embryo formation rate is about 60%), the callus that will have somatic embryo is transferred to somatic embryo is sprouted and callus continuation differentiation and bud formation (Fig. 4).

(4) taking root and transplanting of regeneration bud: the Festuca Arundinacea regeneration bud generally all has the formation (Fig. 5) of root in differentiation.Not excessive bud length forwards it to during to about 1cm cultivates the 14d root and can reach 3cm on the 1/2 MS medium, can carry out the transplanting of regrowth this moment.Regrowth is transplanted to and is placed in the flowerpot that (humidity is 60%～80% in the climatic cabinate, temperature (25 ± 1) ℃, photoperiod is the dark 10h of illumination 14h/) cultivate and two weeks can be transplanted to the land for growing field crops, transplanting survival rate can reach 100% (Fig. 6) under the situation of fungal disease not having.

All medium in this experiment all are transferred to 5.8 with pH before sterilization, sterilising conditions is 121 ℃ of following 25min.Culturing room's temperature is (25 ± 1) ℃.Photoperiod is the dark 10h of illumination 14h/.The intensity of illumination on culture surface is about 3000lx.

Description of drawings

Fig. 1: the growth conditions of callus on proliferated culture medium.

Fig. 2: callus is the organizator blast in enrichment culture.

Fig. 3: somatic embryo forms on the somatic embryo inducement medium in a large number.

Fig. 4: callus is differentiation and bud formation on differential medium.

Fig. 5: regeneration bud continued growth and the formation of root is arranged on differential medium.

Fig. 6: the Festuca Arundinacea regrowth is transplanted in the flowerpot and is survived.

Embodiment

Following embodiment is in order to explain the present invention in more detail, to be confined to this but should not be construed as the present invention.

Experiment reagent:

The mineral salt of using in this experiment are Liu Lidian chemical plant, Beijing product, 2,4-D, KT and caseinhydrolysate are SIGMA company product, vitamin is Beijing fragrant grass pharmaceutical ﹠ chemicall research development corporation product, and sucrose is Beijing northization fine chemicals Co., Ltd product.

Embodiment 1

With U.S. Festuca Arundinacea cultivar ' surpro ' is the examination material, purchases the lawn research institute in Chinese agricultural university.Choose 150 of full seeds, be used in 75% alcohol after shelling and soak 3min, the back then, is thoroughly washed 5 times with sterile distilled water with 0.1% the mercury chloride 10min that sterilizes.The seed that disinfects is inoculated into callus of induce medium MSM and contains 5mg/L, 7mg/L, and 9mg/L 2, carry out callus induction under three processing of 4-D and 0.025mg/L KT (each handles 50 seeds) and cultivate.Induce the callus of formation all to be transferred to MSM down each processing after 2 weeks and contain 4.5mg/L2, the callus of 4-D and 0.2mg/L KT keeps breeding on the medium.The callus enrichment culture is after 3 weeks, is divided into the mung bean size and transfers to MSM and contain 2mg/L 2, carries out the inducing culture of somatic embryo on the medium of 4-D and 0.2mg/L KT (caseinhydrolysate content is 1mg/L), and each blake bottle is placed 5 callus.The callus that will have somatic embryo after 2 weeks is inoculated on the differential medium MSH+2mg/L KT+2% sucrose (caseinhydrolysate content is 2mg/L) and makes its differentiation and bud formation.Differentiation culture will break up ready-made regeneration bud and forward to and carry out culture of rootage on the 1/2MS medium after 3 weeks, culture of rootage can be transplanted to flowerpot after 2 weeks.The differentiation of calli rate that statistics obtains is 93%, 31 strains of emerging of average every callus.

Embodiment 2

With U.S. Festuca Arundinacea cultivar ' Coronado ' is the examination material, purchases the lawn research institute in Chinese agricultural university.Choose 150 of full seeds, be used in 75% alcohol after shelling and soak 3min, the back then, is thoroughly washed 5 times with sterile distilled water with 0.1% the mercury chloride 10min that sterilizes.The seed that disinfects is inoculated into callus of induce medium MSM and contains 6mg/L, 8mg/L, and 10mg/L 2, carry out callus induction under three processing of 4-D and 0.025mg/L KT (each handles 50 seeds) and cultivate.Induce the callus of formation all to be transferred to MSM down each processing after 2 weeks and contain 4.5mg/L2, the callus of 4-D and 0.2mg/L KT (caseinhydrolysate content is 2mg/L) keeps breeding on the medium.The callus enrichment culture is after 4 weeks, and the callus that will have somatic embryo is inoculated on the differential medium MSH+2mg/L KT+2% sucrose (caseinhydrolysate content is 2mg/L) and makes its differentiation and bud formation.Differentiation culture is after 4 weeks, and the regeneration shoot root that is differentiated to form reaches 3cm, is transplanted to flowerpot.The differentiation of calli rate that statistics obtains is 91%, 26 strains of emerging of average every callus.

Embodiment 3

' Summer Lawn ' is the examination material, purchases the lawn research institute in Chinese agricultural university with European cultivar.Choose 150 of full seeds, be used in 75% alcohol after shelling and soak 3min, the back then, is thoroughly washed 5 times with sterile distilled water with 0.1% the mercury chloride 10min that sterilizes.The seed that disinfects is inoculated into callus of induce medium MSM and contains 5mg/L, 7mg/L, and 9mg/L 2, carry out callus induction under three processing of 4-D and 0.025 mg/L KT (each handles 50 seeds) and cultivate.Induce the callus of formation all to be transferred to MSM down each processing after 2 weeks and contain 4.5mg/L2, the callus of 4-D and 0.2mg/L KT (caseinhydrolysate content is 1mg/L) keeps breeding on the medium.The callus enrichment culture was transferred to MSM with it and is contained 2mg/L 2 after 3 weeks, carried out the inducing culture of somatic embryo on the medium of 4-D and 0.1mg/L KT (caseinhydrolysate content is 1mg/L).The callus that will have somatic embryo after 2 weeks is inoculated on the differential medium MSH+2mg/LKT+2% sucrose (caseinhydrolysate content is 1mg/L) and makes its differentiation and bud formation.4 week of differentiation culture back regeneration shoot root reaches 3cm, and it is transplanted to flowerpot.The differentiation of calli rate that statistics obtains is 89%, 24 strains of emerging of average every callus.

Embodiment 4

With U.S. Festuca Arundinacea cultivar ' Fawn ' is the examination material, purchases the lawn research institute in Chinese agricultural university.Choose 150 of full seeds, be used in 75% alcohol after shelling and soak 3min, the back then, is thoroughly washed 5 times with sterile distilled water with 0.1% the mercury chloride 10min that sterilizes.The seed that disinfects is inoculated into callus of induce medium MSM and contains 5mg/L, 7mg/L, 9mg/L 2, carrying out callus induction under three processing of 4-D and 0.025mg/L KT (each handles 50 seeds) cultivates, induce the callus of formation all to be transferred to MSM down each processing after 2 weeks and contain 4.5mg/L2, the callus of 4-D and 0.2mg/L KT keeps breeding on the medium.The callus enrichment culture was transferred to MSM with it and is contained 2mg/L 2 after 3 weeks, carried out the inducing culture of somatic embryo on the medium of 4-D and 0.1mg/L KT.The callus that will have somatic embryo after 2 weeks is inoculated on differential medium MSH+2mg/L KT+2% (caseinhydrolysate content the is 2mg/L) sucrose and makes its differentiation and bud formation.4 week of differentiation culture back regeneration shoot root reaches 3cm, can be transplanted to flowerpot.The differentiation of calli rate that statistics obtains is 96%, 33 strains of emerging of average every callus.

Embodiment 5

With U.S. Festuca Arundinacea cultivar ' Fawn ', ' Summer Lawn ' purchases the lawn research institute in Chinese agricultural university for the examination material to European cultivar.Respectively choose 150 of two kind full seeds, be used in 75% alcohol after shelling and soak 3min, the back then, is thoroughly washed 5 times with sterile distilled water with 0.1% the mercury chloride 10min that sterilizes.The seed that disinfects is inoculated into the additional 9mg/L of callus of induce medium MS mineral salt, 2, (caseinhydrolysate and vitamin B group are respectively 0.5 of component in the MSM medium for 4-D and 0.025mg/L KT, 1.0,1.5 carrying out callus induction under three processing doubly) (each handles 50 seeds) cultivates, induce the callus of formation all to be transferred to MSM down each processing after 2 weeks and contain 4.5mg/L 2, the callus of 4-D and 0.2mg/L KT keeps breeding (organic component keeps self-consistentency in same processing) on the medium.The callus enrichment culture is after 3 weeks, the callus that two kind tall fescue seeds are formed is corresponding respectively transfers to the inducing culture that carries out somatic embryo on MS mineral salt and the organic component content somatic embryo inducement medium (containing 2mg/L2,4-D and 0.1mg/L KT) identical with previous step.The callus that will have somatic embryo after 2 weeks is inoculated into and makes its differentiation and bud formation (identical in same processing series caseinhydrolysate and vitamin B group content and callus of induce, maintenance and the somatic embryo inducement on the differential medium MSH+2mg/L KT+2% sucrose, promptly 0.5,1.0, the amount of 1.5 times of component caseinhydrolysate and vitamin B group in the MSM medium).4 week of differentiation culture back regeneration shoot root reaches 3cm, is transplanted to flowerpot.Statistics obtains ' Fawn ' and ' the differentiation of calli rate of Summer Lawn ' two kinds is respectively 91% and 90%, and the number of emerging of average every callus is respectively 32 strains and 28 strains (mean values under three processing of same kind).

To sum up, in the present invention the callus induction of Festuca Arundinacea can one comparatively wide by 2, (5～10mg/L) carry out, and callus of induce is not the key of regeneration frequency in the 4-D scope.Because callus can expand numerous in a large number, can not cause harmful effect to subsequent experimental even the callus of induce rate is low in enrichment culture yet.The present invention tries that the callus of induce rate is up to 92% in each kind, and minimum is 22%.2,4-D is the key that callus induction forms, and is general 2, callus of induce rate height when 4-D concentration is low, but early stage, the callus growth was slow; 2, the corresponding decline of callus of induce rate when 4-D concentration is high keeps growing rapidly on the medium but be transferred to callus.Add the quality that a small amount of KT can improve callus in the inducing of callus, add the KT of 0.025mg/L in this experiment, raising callus quality plays good effect in callus induction.Can be sure of to add other micro-cytokinin-like substance and also can in the inducing of callus, play desirable effect.Somatic embryo induce the number that sprouts that can increase differentiation of calli rate and unit callus.Organic principle in the medium changes slightly influences result of experiment not too largely.

Claims (5)

1. one kind obtains the method for a large amount of Festuca Arundinacea regeneration plants by tissue culture, and this method comprises that (1) select explant; (2) at MSM medium supplemented 5～10mg/L 2, carry out callus induction on the callus of induce medium of 4-D and 0.025mg/L kinetin, and after described callus formed for two weeks, it is transferred to subculture medium MSM+4.5mg/L 2, carry out enrichment culture on 4-D+0.1～0.2mg/L KT; (3) on callus differential medium MSH+2mg/L KT+1%～2% sucrose, break up callus; (4) transplant regeneration bud;

The basis of described MSM medium is the mineral salt of MS medium, additional 2mg/L caseinhydrolysate, 9.9mg/L VB ₁, 9.5mg/L VB ₆, 4.5mg/L niacin, 8g/L agar and 30g/L sucrose;

The composition of described MSH medium is the molysite of the macroelement of SH medium+MS medium and trace element+caseinhydrolysate 1-2mg/L+VB ₁9.9mg/L+VB ₆9.5mg/L+ niacin 4.5mg/L+ agar 8g/L+1%-2% sucrose.

2. the process of claim 1 wherein that described explant is the full mature seed of Festuca Arundinacea.

3. the method for claim 1 is characterized in that carrying out carrying out inducing of somatic embryo earlier before the described callus differentiation.

4. the method for claim 3, inducing of wherein said somatic embryo is at medium MSM+2mg/L 2, carries out on 4-D+0.1～0.2mg/L KT, wherein concentration of sucrose is 3%.

5. a callus differential medium that is used to obtain a large amount of Festuca Arundinacea regeneration plants is specially: MSH medium+2mg/L KT+1%～2% sucrose;

The composition of described MSH medium is the molysite of the macroelement of SH medium+MS medium and trace element+caseinhydrolysate 1-2mg/L+VB ₁9.9mg/L+VB ₆9.5mg/L+ niacin 4.5mg/L+ agar 8g/L+1%-2% sucrose.

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