CN1724654A - Establishent of acacia crassicarpa high efficiency transforming system induced by agricultural bacillus - Google Patents
Establishent of acacia crassicarpa high efficiency transforming system induced by agricultural bacillus Download PDFInfo
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Abstract
The invention builds high efficiency rebirth system generated through organic of Acacia crassicarpa. The system uses the external plant body as inducement rebirth material to successfully rebirth a whole plant. Through adding different group compounding thickness plant growth adjusting agent into improved MS cultivate medium, under the best condition, mature phyllode would induce callus at the 10 days, and indefinite bud at about the 40 days. The ratio that the callus would polarize indefinite bud is 56.25%. The indefinite bud could successfully plant to earth after growth and rooting. The technology is easy, high efficiency and easy to repeat.
Description
One, technical field
The present invention relates to plant genetic engineering field.Be specifically related to the foundation of acacia rachii high efficient regeneration system and agriculture bacillus mediated acacia rachii transformation system of high efficiency.
Two, background technology
It is important genus in the pulse family (Mimosoideae) that yearning between lovers belongs to (Acacia), comprises about 1200 kinds, abounds with in Australia Africa, India and America
[3](Simmons1987; Jones et al.1990), it is a kind of to originate in the only Acacia confusa (A.confusa) of China.Most of yearning between lovers can be produced first-class firewood, and some are arranged then is to produce paper pulp, rubber, protein, tannin, coating, the good resource of ink and flavouring agent.Acacia rachii is that the good short wheel of a class cuts down the planting out seeds, adaptability is strong, speed is given birth to, be suitable for the torrid zone, subtropics growth and breeding, particularly outstanding is root system and the root nodule bacterium symbiosis of acacia rachii, fixed nitrogen is improved the soil rapidly, and huge value (Palmberg 1981) is arranged in artificial afforestration and wasteland reclamation.Yearning between lovers can also be detected soil corrosion effectively, and helps dikaka.
Well-known is that the regeneration rate of pulse family trees in its physical environment is lower.The expansion cultivation of Acacia crassicarpa needs the clonal propagation of choiceness and external efficiently breeding technology thereof, comprises little numerous and regeneration techniques.In addition, biotechnology such as genetic transformation, somatic cell clone are selected, monoploid produces also needs reliably, regeneration system rapidly efficiently.Though yearning between lovers seeds regeneration difficulty, and variety of issue (Nangia and Singh can appear under its condition of in vitro culture, 1996), but in recent years, bibliographical information the regeneration of several acacia rachiis, comprise catechu A.catechu (.1995 such as Rout), acacia auriculiformis A.auriculiformis (Rao and Prasad 1991), horse accounts for yearning between lovers A.Mangium, and (Ahmad 1991; Bhaskar and Subhash 1996; .1991[such as Galiana have only one piece] a, b; Xie and Hong 2001a, b), Sinuate Acacia A.sinuata (.2000 such as Vengadesan .2002 such as Vengadesan), gum arabic Acacia A.nilotica (.1996 such as Garg) and lappa A.farnesiana (.2000 such as Ortiz), but regeneration rate is all lower.Yet the foundation of relevant thick-walled pod yearning between lovers regeneration system rapidly does not but appear in the newspapers the report that its bud organ of bibliographical information, rudiment bar is just arranged and have the tender stem micropropagation of terminal bud.
The external breeding technology of trees comprises micropropagation, and organ takes place, somaclonal variation, and mutagenesis and somatic embryo take place.Micropropagation is the important channel of external breeding in the forestry biotechnology, this technology can save time and the spatial condition under produce millions of individuals with same.The conventional propagation method of acacia rachii is based on seminal propagation, because the yearning between lovers seed has certain branch voltinism, The Characters differences such as it is tree-like, increment, resistance, fibre content are big, retrained their development and use to a certain extent.The success of yearning between lovers seeds micropropagation makes these problems be readily solved.At present, there are more than the 20 kind of yearning between lovers seeds of reaching to use wild explant to carry out more general micropropagation, as sting leaf Acacia (A.senegal), gum arabic Acacia (A.nilotica), acacia auriculiformis (A.auriculiformis), Sinuate Acacia (A.sinuata) and horse account for yearning between lovers (A.mangium), thick-walled pod yearning between lovers (A.crassicarpa), volume pod yearning between lovers (A.cincinuata), hybridize yearning between lovers tabernaemontanus bulrush screen No. one (A.mangium * A.auriculiformis) etc.
The isolated organ of plant is meant that stripped tissue or cell form the process of organs such as indefinite bud, root and bud under the condition of tissue culture.Isolated organ is one of topmost mode of plant regeneration whole plant.It not only has important use value on a large amount of asexually propagated plants, and is to utilize biotechnology plant to be carried out the prerequisite and the basis of genetic improvement.In some biotechnologys, cultivate somatic cell hybrid, utilize pollen and endosperm acquisition monoploid or polyploid plant and cultivation transgenic plant or the like all to need to carry out isolated organ generation and plant regeneration as cytogamy.Organ takes place generally to be subjected to inducing of exogenous plant hormones level, and directly takes place from explantation tissue or by callus.In acacia auriculiformis and Ke A Acacia, the stem apex explant that takes off from the seedling of land for growing field crops and asepsis growth can produce indefinite bud through the callus stage.In the tissue culture of Acacia confusa, cotyledon is considered to best explant.Also the someone attempted from well differentiated tissue, such as form layers and phloem, inducing callus in thorn leaf Acacia.The stem apex of Ken Shi yearning between lovers (A.cuninghamia), tender stem section and blade also can successfully induce regeneration bud
[62]Other explant also comprises the gum arabic Acacia, the hypocotyl of Sinuate Acacia and turn-knob yearning between lovers, and the ripe cotyledon of catechu and Sinuate Acacia, horse accounts for the cotyledon of yearning between lovers, cotyledonary node, plumular axis, leaflet, petiole, the thin cellular layer of stem and young stem.In inducing organogenetic process, use the combination of plant hormone and phytokinin mostly.The order that plant hormone is selected is 2,4-D＞NAA＞IAA＞IBA.In Ke Ah Acacia, acacia auriculiformis, Sinuate Acacia and horse account for yearning between lovers, Acacia confusa, in the adventitious bud inducing of Ken Shi yearning between lovers, 6-BA is the most frequently used phytokinin.In the organ generating process of acacia rachii, it is generally acknowledged that BA can increase the tightness degree of callus.Also there is research report to point out, uses TDZ to account for successful evoking adventive bud in the callus of yearning between lovers separately horse.Also have bibliographical information, KT also can be used for acacia auriculiformis, and the organ of catechu and Sinuate Acacia takes place.
In most yearning between lovers seeds, multiple type of culture medium can cause regeneration.Wherein the MS substratum is the most commonly used, and the 1/2MS success also can be used for the regeneration of Acacia Acacia sinuate.With MS substratum and 1/2MS substratum Ke Ah Acacia is carried out regeneration test, found that the MS substratum can induce more indefinite bud.In catechu yearning between lovers, WPM and 1/2WPM also are used for the regeneration induction bud by success.Recently, Douglas (2000) and McNamara find that horse accounts for yearning between lovers and can carry out the regeneration of indefinite bud in the DKW substratum.For Ke Ah Acacia, Schenk and Hildebrandt substratum both can be used for callus induction, can be used for the regeneration of indefinite bud again.
Somatic embryo derive from one unicellular, take place such as organ with those and cellulous breeding such as micropropagation is different.It is a cell the strongest totipotent proof, has reported the somatic embryo generation in a lot of woody trees, and comparatively difficulty takes place woody fabaceous somatic embryo.In many yearning between lovers seeds, explant jejune and young age often is used to induce embryo callus subculture.Immature cotyledon, immature endosperm and immature zygotic embryo are used for respectively at catechu, the gum arabic Acacia, lappa, horse accounts for yearning between lovers, and the inductor cell stage takes place and the acquisition seedling in the acacia auriculiformis.And Sinuate Acacia carries out inducing of somatic embryo generation with leaf, and all as a result callus all generate the embryo and develop into plant.As explant inducing embryo in thorn leaf Acacia success takes place also to have obtained with cotyledon.In the somatic embryo of acacia rachii took place, the NAA that is to use of report was obtained by suspension culture by immature cotyledon in catechu at first.Use NAA and KT to generate callus and somatic embryo (globular embryo, heart, torpedo shape).Only in not adding the 1/2MS substratum of growth regulator, embryo could be sprouted.The somatic embryo that NAA that Das working concentrations such as (1996) is lower and KT have induced catechu, only the embryo could be taken place under the condition that NAA exists.
Obtain in the process of embryo callus at acacia auriculiformis, functions of hormones particularly 2, the effect of 4-D is the most key.The immature endosperm of gum arabic Acacia is at 2 of 2mg/L, and the BA of 4-D and 5mg/L induces down and produced embryo callus subculture.The dark cultivation increased the frequency that the embryo generates.Inducing in the embryo callus subculture by the immature zygotic embryo of lappa and Sinuate Acacia, 2,4-D also plays an important role, and can obtain different embryo stages in the MS minimum medium.Xie and Hong (2001.b) use TDZ and IAA to induce embryo callus subculture recently.Use GA
3Can obtain the torpedo-shape embryo and the embryo in cotyledon stage.Shahana and Gupta (2000) use ZT (2mg/L) directly to obtain embryo's generation by the cotyledon of thorn leaf Acacia.Add other plant hormone, as 2,4-D and NAA, can only generate a spot of embryo, particularly NAA has also increased the probability that abnormal embryo occurs.Although the ripening stage can take root, the aplasia of plumule leaf.Removing thorn leaf Acacia will be bred by the form layers tissue in the KB substratum, and the cells,primordial of acacia auriculiformis is suitable for obtaining on B5 medium.In most of callus inductions and regenerative process, WPM and MS substratum can use.WPM, MS and general MS substratum are used in the generation of somatic embryo.And sprout and maturation, account for yearning between lovers horse, can use MS minimum medium or 1/2MS substratum in thorn leaf Acacia and the lappa.
The forest traditional breeding method seed selection time is long, efficient is low.Genetically engineered is that a tempting new way has been opened up in forest genetics, not only can shorten breeding cycle widely, can transform forest genetics of resistance material again on gene level, has more science and accuracy, has improved the purpose and the operability of breeding.In addition, also enlarge the scope of breeding, broken the dysgenesia between the species, realized the common point of gene.Along with the foundation of various transformation systems and progressively perfect, and the going deep into of forest genetically engineered fundamental research, from now on to forest purpose is arranged, genetic improvement can be more prone to targetedly.The research of transgenosis forest starts from the eighties in 20th century, and the first transgenosis forest was succeedd on willow in 1986.For over ten years, this biotechnology of transgenosis is very rapid at the application development that improves aspects such as forest resistance, improvement quality, technical being gradually improved.
At present, the research of transgenosis acacia rachii also is in the starting stage, and the research of relevant transgenosis acacia rachii both at home and abroad only limits to several pieces of odd reports.Obtain complete transfer-gen plant and need 3 key elements: set up regeneration system rapidly efficiently; Structure carries the suitable carrier system of foreign gene; Set up gene transformation system efficiently.Wherein the foundation of high efficient regeneration system is the important prerequisite that successfully obtains the transgenosis acacia rachii.At present, the regeneration rate of the acacia rachii of having reported is all lower, no matter adopts which kind of transgenic technology, improves its transformation efficiency and seems particularly crucial.With regard to transformation technology, particle bombardment transform have the restriction that is not subjected to transformation receptor, simple to operate, advantage such as can regulate, but be difficult to control genetically modified copy number, the workload of screening is bigger.Agriculture bacillus mediated genetic transformation is existing successful report aspect the acacia rachii transgenosis.Xie D Y, Hong Y (2002) infects the stem section that horse accounts for yearning between lovers body vitro culture with the agrobacterium tumefaciens lba4404 that contains plasmid pBI121, has obtained to change the regeneration plant of NPTII gene.
Because the regeneration system rapidly regeneration rate of acacia rachii is lower in the prior art, genetically modified transformation efficiency is also lower, therefore, is necessary to develop the high efficient regeneration system of a kind of acacia rachii, and utilize that this system carries out by agriculture bacillus mediated transformation system of high efficiency.
Three, summary of the invention
An object of the present invention is to provide the high efficient regeneration system of acacia rachii.Another object of the present invention provides agriculture bacillus mediated acacia rachii transformation system of high efficiency.Another object of the present invention provides the external propagation method of the acacia rachii that utilizes described high efficient regeneration system.
Technical problem to be solved by this invention is: the regeneration system rapidly regeneration rate of acacia rachii is lower in the prior art; Genetically modified transformation efficiency is also lower; External propagation method is very complicated relatively, and efficient is not high.
The present invention implements by the following technical programs:
1. pass through suitable medium, preferred MS substratum, carry out the cultivation of acacia rachii seed, select acacia rachii cotyledon, hypocotyl, internode, sophisticated phyllodium etc. as explant, the regeneration of evoking adventive bud under different hormone combinations is tested the high efficient regeneration system of determining acacia rachii through the transplanting of the elongation of indefinite bud, the seedling of taking root and take root.
2. on suitable medium, cultivate the Agrobacterium that contains the purpose expression vector, transform above-mentioned high efficient regeneration system by routine techniques, promptly be inoculated in the acacia rachii young leaflet tablet explants such as (also claiming phyllodium) of cotyledon, hypocotyl and isolated culture or greenhouse growth, determine agriculture bacillus mediated acacia rachii transformation system of high efficiency through succeeding transfer culture, root induction and transplanting test.
Technique effect of the present invention is as follows:
1. set up the high efficient regeneration system of acacia rachii, this system forms fairly simple, and it is shorter to set up required time, and the regeneration efficiency height easily repeats.Utilize this system, promptly induced callus (joint knot) in 10 days, induced indefinite bud in 40 days, warty callus (joint knot) differentiates the ratio of indefinite bud up to 56.25%, indefinite bud is through cultivating, and rooting rate reaches 100%, and the plantlet of taking root is through domestication, can successfully transplant in soil, the growth phenotype of regeneration plant is normal.
2. utilize above-mentioned high efficient regeneration system, set up the Agrobacterium tumefaciens mediated transgenosis system of soil, can successfully foreign gene be imported in the plant by this method, this system's transgene efficiency height, and can carry out fairly large conversion.
Four, description of drawings
Fig. 1 is warty callus figure.
Fig. 2 is a large amount of indefinite bud figure that warty callus surface takes place.
Fig. 3 is the stretch scheme of indefinite bud.
The lateral root figure of Fig. 4 for producing on the indefinite bud.
The plant figure that Fig. 5 obtains through hardening and transplanting for indefinite bud.
Fig. 6 is the resistance warty callus figure through Agrobacterium-mediated Transformation.
Fig. 7 is a large amount of indefinite bud figure that resistance warty callus surface takes place.
Fig. 8,9 stretch schemes for screening culture medium inductive indefinite bud through containing 0.1mg/l TDZ.
Figure 10 is the figure of taking root of the indefinite bud through transforming.
The PCR detected result that Figure 11 transforms for foreign gene 4CLI.The swimming lane both sides are 2000bp standard molecular weight Marker; Negative and the positive control of CK-and CK+; 1-10 is the result of 4CLI gene PCR amplification in plants transformed.
Five, embodiment
Following specific embodiment will be made explanations and illustrate the present invention, but protection scope of the present invention is not subjected to the restriction of specific embodiment.
Material therefor is commercially available getting in the following example.
The abbreviation of relational language is as follows: BA 6-benzyladenine CM coconut palm breast GA3 gibberic acid IBA 3-indolebutyric acid KT kinetin MS Murashige and Skoog (1962) mediumNAA α-Nai Yisuan IAA indole-3-acetic acid TDZ1-phenyl-3-(1,2,3-thiadiazol-5-yl) Ureathidiazuron) 2, the 4-D 2,4 dichlorophenoxyacetic acid
The high efficient regeneration system of embodiment 1 Acacia crassicarpa
1.1 vegetable material with disinfect
Sophisticated seed was handled 1 minute in 100 ℃ of water, soaked in distilled water 16 hours again, used 70% ethanol surface sterilization then 5 minutes, again in 0.1% HgCl
2In (adding several soil temperatures 20) sterilization 1 minute, with aseptic water washing 3-4 time, the disinfectant seed (contains 0.4% agar) again on 1/2MS substratum sprouting.
1.2 the preparation of substratum and culture condition
Substratum was 121 ℃ of sterilizations 15 minutes.Medium pH to 5.8 is adjusted with NaOH (aseptic) solution of 1N in the sterilization back.Plant-growth regulator carries out filtration sterilization with the filter membrane of 0.2 μ m.Unless otherwise indicated, culturing room's temperature is 28 ℃, and (intensity of illumination is 26 μ mols under illumination condition
-1m
-2) cultivate illumination every day 16h.
1.3 inducing of warty callus and indefinite bud
Seed is through being inoculated in after the surface sterilization on 1/2 intensity, the sugar-free MS substratum (containing 0.4% agar), and germinates in 10-12 days.Get phyllodium from the seed seedling in 60 day age, be cut into 0.3 * 0.5-cm size as explant.Explant is cultivated 10 days----TDZ and the combination of NAA different concns containing on the substratum of different hormone combinations; KT and 2, the combination of 4-D different concns; KT and the combination of NAA different concns; BA and the combination of IAA different concns; And BA and 2, the combination of 4-D different concns.Each processing comprises 100 explants, repeats twice.All substratum all add the coconut palm breast of 5% (v/v), the sucrose of 30g/l, and the agar of 0.4% (w/v).The inductivity of statistics green or purple green warty callus.
Cultivate after 10 days, the callus subculture continues forming evoking adventive bud on the identical substratum.Each processing comprises 100 callus, repeats twice.Per 10 days subcultures once.Raw data is record after cultivating 40 days, and this moment, existing macroscopic indefinite bud took place.
1.4 the elongation of indefinite bud and taking root
The indefinite bud clump that has pinnately compound leaf that induces from the substratum that contains 0.5mg/l TDZ and 0.5mg/l NAA is used to carry out next step test.Indefinite bud is inoculated in and contains GA
3(0,0.5,1mg/l) and TDZ (0, on basic MS culture medium 0.1mg/l), carry out bimestrial cultivation.All substratum all add the coconut palm breast of 5% (v/v), the sucrose of 30g/l, and the agar of 0.4% (w/v).Downcut the long bud of 2-3cm and be inoculated in the 1/2 MS substratum that contains 0.5mg/l IBA, take root.
The transplanting of seedling 1.5 take root
Root culture is after hardening, and the aseptic seedling that will have at least 15 lateral roots is taken out.Gentle flushing under tap water, the substratum of the clean seedling base portion of taking root.Be transplanted in the matrix of sterilization, matrix contains sand, vermiculite, garden mould 0.5: 0.5: 1 (v/v).Keep 80% relative humidity, intensity of illumination is 26 μ mols
-1m
-2, illumination every day 16h.Watered one time water in per three days.
1.6 result
1.6.1 warty callus of induce
The warty callus betides the pulsating incision of phyllodium at first.The substratum of all kinds all can induce warty callus (see figure 1), and the difference on effect between the different sorts substratum is not obvious.
1.6.2 inducing of indefinite bud
The combination of TDZ and NAA, the generation of indefinite bud has successfully been induced in the combination of BA and IAA.Through 40 days cultivation, a large amount of indefinite bud (see figure 2)s took place in the surface of warty callus.
The various combination evoking adventive bud regeneration rate from 1.6% to 56.25% of TDZ and NAA does not wait.0.5mg/l the combination regeneration rate of TDZ and 0.5mg/l NAA reaches 56.25%.And the regeneration rate of 3mg/l BA and 0.6mg/l IAA combination is lower than 5%.Other hormone combinations have only produced secondary warty callus.
1.6.3 the elongation of indefinite bud with take root
The substratum that contains 0.1mg/l TDZ has effectively been induced the elongation (see figure 3) of indefinite bud.Be transferred to after the elongation on the 1/2MS substratum that contains 0.5mg/l IBA and take root.Nearly all indefinite bud has all produced 10 lateral root (see figure 4)s in one month.Hardening and transplanting have also obtained successful (see figure 5).
As seen, set up one than advantages of simplicity and high efficiency thick-walled pod yearning between lovers (A.crassicarpa.) by organogenetic regeneration system, successfully regenerate by organogenetic mode with ripe thick-walled pod yearning between lovers phyllodium.
The Acacia crassicarpa transformation system of high efficiency that embodiment 2 is agriculture bacillus mediated
2.1 the cultivation of Agrobacterium
(1) (be Kan 100mg/L containing suitable microbiotic, Str100mg/L) on the YEB solid medium, containing the Agrobacterium Agrobacterium tumefaciensLBA4404 bacterial strain line coated plate that inserts the purpose plant expression vector, in 28 ℃ of cultivations 48 hours, contain the 4CLI gene of willow in this purpose plant expression vector;
(2) select activatory list bacterium colony in the step 1, be inoculated in YEB (pH7.0) liquid nutrient medium of 2ml, contain in this liquid nutrient medium with step 1 in the microbiotic of same composition and concentration (be Kan100mg/L, Str 100mg/L), and under 28 ℃, 220rpm condition, shaking incubated overnight on the shaking table;
(3) take out the Agrobacterium of shaking incubated overnight, dilute in YEB (pH7.0) liquid nutrient medium that is inoculated in 20-50ml (consumption of substratum depends on the quantity of plant explant to be transformed), must add in this liquid nutrient medium with step 1 in the microbiotic of same composition and concentration (be Kan100mg/L, Str 100mg/L), and add a spot of Syringylethanone (AS), under 28 ℃, 220rpm condition, shaking incubated overnight on the shaking table;
(4) Agrobacterium of the above-mentioned incubated overnight of low temperature (4 ℃) centrifugal (10000rpm, 5 minutes) abandons supernatant, and collecting precipitation precipitates the MS liquid nutrient medium diluted for use with 1/2.
2.2 Agrobacterium-mediated Transformation acacia rachii
The acacia rachii young leaflet tablet (also claiming phyllodium) of cotyledon, hypocotyl and isolated culture or greenhouse growth all can be used as the explant of inoculation usefulness, and the preparation of explant and inoculation method carry out (Ye Panfa of improvement) by follow procedure:
(1) cotyledon and hypocotyl transform: downcut young tender cotyledon and hypocotyl from the aseptic seedling of sprouting 7-10 days, each cotyledon cuts in half and cuts blade tip, it is long that hypocotyl is cut into 0.5cm, place the Agrobacterium diluent that fills step 4 preparation then immediately, immerged time is 10-15 minute, and moving with the have gentle hands jog;
(2) mature leaf (phyllodium) transforms: mature leaf can be taken from and sprout about 60 days aseptic seedling; Also can take from the plant of hot-house culture, to hot-house culture plant as conversion, should spray the sterilant of an amount of concentration weekly, use the commercially available thimerosal of 20% (v/v) or 5% clorox surface sterilization (and adding several soil temperatures 20) 10 minutes before the conversion again, then for several times with aseptic water washing.In some cases, also can use the mercury chloride of 0.05-0.1 to carry out disinfection, but all influential to the vitality and the transformation efficiency of phyllodium.And then aseptic phyllodium is cut into the square (and vertical make some wounds gently) of 5-7 square millimeter along vein with sterile razor blade with vein, the leaf dish that newly prepares is invaded rapidly in the Agrobacterium diluent of step 4 preparation, immerged time is 10-15 minute, and moving with the have gentle hands jog;
(3) take out the explant that transforms as stated above with tweezers rapidly, place on the aseptic filter paper with absorption of residual and stay agrobacterium liquid on the explant, and then explant is inverted in not contain cultivates 2-3 days (25 ℃) on any antibiotic regeneration culture medium under the dark condition altogether, Agrobacterium is successfully contaminated infect blade;
2.3 transform the cultivation of explant
(1) explant after the dark cultivation of taking-up is used aseptic water washing explant 3 times, removes the Agrobacterium that is adsorbed on the explant surface.If the explant surface growth has too much Agrobacterium, can in sterilized water, add the Pyocianil of 500mg/L, wash 3 times, be adsorbed on the too much Agrobacterium in explant surface to kill to remove.And then explant placed on the selective medium that contains antibiotic in 28 ℃ of following illumination cultivation, to select cell transformed, the regeneration culture medium that consists of of resistance substratum adds the kantlex (Kan) of 20mg/L and the Pyocianil (Cb) of 500mg/L;
(2) succeeding transfer culture: for the first time the time of succeeding transfer culture is that carry out after the step 6 7-10 days, and continuing on the selective medium of transferring to new preparation through the explant that transforms to select to cultivate, the prescription of selective medium is formed as step 6.Later succeeding transfer culture serves as to carry out at interval with per two weeks, culture condition such as step 6, and the prescription of selective medium is formed as step
6, till callus forms indefinite bud;
(3) be transferred to the budlet elongation of cultivating on the selectivity elongation medium on the callus that makes conversion at the callus that forms indefinite bud on the selective medium, and kill the false positive plant, the selectivity elongation medium consist of MS+3% sucrose+5% coconut palm breast+0.1mg/lTDZ+300mg/LCar+20mg/Lkan, this process will continue effect in 2 months, and succeeding transfer culture cycle and culture condition are with step 6;
(4) resistant buds about length 2cm is downcut, place root induction on the selectivity root media, the selectivity root media consist of 1/2MS+3% sucrose+5% coconut palm breast+0.5mg/L IBA+300mg/LCar+20mg/LKan, succeeding transfer culture cycle and culture condition are with step 6,2-3 has root to occur after week, but this process generally needs a wheat harvesting period.
2.4 checking
(1) gets the blade of 1g effect from the transformed plant of having taken root, extract plant genome DNA, by the integration situation of PCR method checking foreign DNA with micromethod;
(2) being transplanted in the soil through the transgenosis acacia rachii of PCR checking, after indoor experienced seedling is about 10 days, place the greenhouse growth again, and get young leaflet tablet further by PCR from the plant of greenhouse growth, Southern, Northern detects.
2.5 result
2.5.1 transform back resistance warty callus
The warty callus betides the pulsating incision of phyllodium at first.(see figure 6)
2.5.2 the acquisition of transformed plant resistance indefinite bud
Through 40 days cultivation, a large amount of indefinite buds took place in the surface of warty callus.(see figure 7)
2.5.3 transformed plant elongation
The screening culture medium that contains 0.1mg/L TDZ has effectively been induced the elongation of indefinite bud, has killed the false positive plant simultaneously.(seeing Fig. 8,9)
2.5.4 transformed plant is taken root
On 1/2MS+3% sucrose+5% coconut palm breast+0.5mg/L IBA+300mg/L Car+20mg/L Km substratum, successfully take root.(see figure 10)
2.5.5 transformed plant PCR detected result
Get 10 young plant PCR and detect the 4CLI gene that transforms, the result proves that the 4CLI gene has successfully changed the acacia rachii plant over to, has successfully made up the acacia rachii transfer-gen plant.(seeing Figure 11)
Utilize the external propagation method of the acacia rachii of described high efficient regeneration system to carry out equally as embodiment 1.
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Claims (9)
1. the high efficient regeneration system of an acacia rachii, comprising: suitable acacia rachii explant comprises acacia rachii cotyledon, hypocotyl, internode, sophisticated phyllodium; Inducing culture, it is for adding the minimum medium of different hormone combinations, and hormone combinations comprises TDZ and NAA, KT and 2,4-D, KT and NAA, BA and IAA, BA and 2,4-D, every kind of hormone concentration is all less than 3mg/l in the combination, and total concn is greater than 0.01mg/l.
2. high efficient regeneration system as claimed in claim 1, wherein explant is 60 days a phyllodium.
3. high efficient regeneration system as claimed in claim 1, wherein minimum medium is the MS substratum.
4. high efficient regeneration system as claimed in claim 1, wherein hormone combinations is TDZ and NAA or 3mg/l BA and 0.6mg/lIAA.
5. high efficient regeneration system as claimed in claim 1, wherein the concentration of hormone combinations TDZ and NAA is respectively 0.01-1.5mg/l and 0-1mg/l.
6. high efficient regeneration system as claimed in claim 5, wherein the concentration of hormone combinations TDZ and NAA is 0.5mg/l.
7. high efficient regeneration system as claimed in claim 1 wherein also comprises indefinite bud elongation medium and root media, and the former is the minimum medium that contains 0.1mg/l TDZ, and the latter is the minimum medium that contains 0.5mg/l IBA.
8. agriculture bacillus mediated acacia rachii transformation system of high efficiency comprises: contain the Agrobacterium of external source goal gene and aforesaid right and require each high efficient regeneration system of 1-7.
9. the external propagation method of an acacia rachii comprises: choose suitable acacia rachii explant, comprise acacia rachii cotyledon, hypocotyl, internode, sophisticated phyllodium; On the MS substratum that contains the combination of 0.5mg/l TDZ and 0.5mg/l NAA plant-growth regulator, induced 10 days, obtain callus, on the MS substratum that contains 0.01-1.5mg/l TDZ and 0-1mg/l NAA, induced 40 days, obtain indefinite bud; Then indefinite bud is transferred to the MS that contains 0.1mg/l TDZ and contain cultivation 2 months; Cultivated 1 month containing on the 1/2 intensity MS of 0.5mg/l IBA through the bud of elongation, take root, transplant and obtain regeneration plant.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007095792A1 (en) * | 2006-02-22 | 2007-08-30 | The Research Institute Of Forestry, Chinese Academy Of Forestry | Method to cultivate transgenic broad-leaf sprouting trees |
| CN103703131A (en) * | 2011-07-25 | 2014-04-02 | 国立大学法人奈良先端科学技术大学院大学 | Novel gene inducing elongation of roots or increasing biomass, and use therefor |
| CN108738777A (en) * | 2018-06-07 | 2018-11-06 | 福建省泉州罗溪国有林场 | A kind of Acacia continma root turion cuttage and seedling culture method |
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| JPS62224224A (en) * | 1986-03-25 | 1987-10-02 | 王子製紙株式会社 | Regeneration of plant body from protoplast of ligneous plant |
| WO2001053452A2 (en) * | 2000-01-19 | 2001-07-26 | Institute Of Molecular Agrobiology | Regeneration and genetic transformation of acacia mangium |
| BR0013482A (en) * | 2000-06-21 | 2002-05-14 | Inst Of Molecular Agrobiology | Somatic embryogenic regeneration of acacia mangium |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007095792A1 (en) * | 2006-02-22 | 2007-08-30 | The Research Institute Of Forestry, Chinese Academy Of Forestry | Method to cultivate transgenic broad-leaf sprouting trees |
| US8076537B2 (en) | 2006-02-22 | 2011-12-13 | The Research Institute Of Forestry, Chinese Academy Of Forestry | Method of breeding germinable transgenic broadleaved tree species |
| CN103703131A (en) * | 2011-07-25 | 2014-04-02 | 国立大学法人奈良先端科学技术大学院大学 | Novel gene inducing elongation of roots or increasing biomass, and use therefor |
| CN103703131B (en) * | 2011-07-25 | 2016-08-17 | 国立大学法人奈良先端科学技术大学院大学 | Induce the elongation of root or the new gene of the amount of increase biomass and application thereof |
| CN108738777A (en) * | 2018-06-07 | 2018-11-06 | 福建省泉州罗溪国有林场 | A kind of Acacia continma root turion cuttage and seedling culture method |
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