CN1582641A - Tetraploid transgenic locust and quick cultivation - Google Patents
Tetraploid transgenic locust and quick cultivation Download PDFInfo
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- CN1582641A CN1582641A CN 200410024208 CN200410024208A CN1582641A CN 1582641 A CN1582641 A CN 1582641A CN 200410024208 CN200410024208 CN 200410024208 CN 200410024208 A CN200410024208 A CN 200410024208A CN 1582641 A CN1582641 A CN 1582641A
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Abstract
A transgenic and tissue culture method for quickly reproducing the tetraploid locust tree includes such steps as sampling the new stem of tetraploid locust tree, disinfecting, start culturing, inducing culture to obtain calli, transgenic treating by Agrobacterium tumefaciens LBA 4404 containing binary carrier pBin 438, differential culturing, selection culturing to obtain resistant plant Kan, molecular testing to obtain transgenic plant, rooting culture, naturalizing and planting in field.
Description
(1) technical field
The present invention relates to the mating system of tetraploid locust, relate in particular to the method for tetraploid locust being carried out tissue-culturing rapid propagation and genetic transformation, belong to plant genetic engineering field or agricultural biological technical field.
(2) background technology
Because the shortage of the shortage, particularly arbor species of anti-salt drought-enduring plant material has seriously restricted northern saline and alkaline arid area ecological engineering construction and rural economic development.Tetraploid locust is the locust tree new varieties that Korea S breeds by chromosome doubling, (strong stress resistance except that the good characteristics that keeps common locust tree, suitable multiple weather and soil condition, timber is hard, and purposes is wide, flower is good nectariferous plant, root has rhizobium, can improve soil), also have good characteristics such as blade is big, protein content is high, tree low bodyization, if can improve its anti-salt drought resistance, significant for saline and alkaline arid area ecology and economic construction.
From separated from spinach in 1981 and partial purification acquisition betaine aldehyde dehydrogenase gene (BADH) after, the BADH gene engineering comes into one's own day by day.The BADH gene that Manabu etc. will derive from barley changes tobacco over to, and under osmotic stress, BADH measures increase, and enzymic activity also is multiplied.Liu Fenghua etc. utilize that the Agrobacterium method is transferred in strawberry and the tobacco, Guo Beihai etc. changes the BADH gene respectively on wheat, the watercress with Li Yinxin etc., and the salt resistance of the transfer-gen plant that obtains also improves.But do not see the conversion report of BADH gene on woody plant so far as yet.About the transgenic technology research of locust tree, Davis etc. carry out the hypocotyl inoculation Agrobacterium tumefaciems (Agrobacterium tumefaciens) of seed seedling Southern to plant DNA and the analysis showed that locust tree is the host plant of Agrobacterium; The Agrobacterium tumefaciems A281 of binary vector pGA472 is carried in inoculation on injured cotyledon, and cotyledon produces the callus of anti-kanamycin as a result, and detection proof NPT II gene order is incorporated on the locust tree genome; Han is inoculated into hypocotyl on the medium that contains agrobacterium rhizogenes R1601, detect to show that T-DNA has been incorporated in the genome, and quiding gene has obtained expression (generations of wrinkle leaf and a large amount of roots).But up to the present, the genes of interest that Shang Weiyou has the production meaning imports locust tree.And tetraploid locust is the report that only limits to carry out with the stem section of axillalry bud tissue culture expanding propagation.
(3) summary of the invention
At above-mentioned deficiency, the problem to be solved in the present invention provides a kind of tetraploid locust agrobacterium co-cultivation mediation genes of interest that carries out and transforms, and carries out the method for the tissue-culturing rapid propagation of transformed plant.
For achieving the above object, technical scheme of the present invention adopts following steps:
(1) tetraploid locust is given birth to then the stem section of young sprout, be seeded in after the sterilization and start in the medium, be transferred in the callus inducing medium after 15～3 days, the callus that obtains, after following Agrobacterium tumefaciems (Agrobacterium tumefaciens) the LBA4404 fungus strain transgenosis that contains binary vector pBin438 is handled, inoculate and form the bud of growing thickly in the differential medium;
Consisting of of above-mentioned MS medium: KNO
31900mgL
-1, NH
4NO
31650mgL
-1, MgSO
4370mgL
-1, KH
2PO
4170mgL
-1, CaCl
22H
20 440mgL
-1, MnSO
44H
2O 22.3mgL
-1, ZnSO
47H
2O 8.6mgL
-1, H
3BO
36.2mgL
-1, KI 0.83mgL
-1, NaMoO
42H
2O0.25mgL
-1, CuSO
45H
2O 0.025mgL
-1, CoCl
26H
2O 0.025mgL
-1, Na
2-EDTA 37.3mgL
-1, FeSO
47H
20 27.8mgL
-1, glycine 2.0mgL
-1, hydrochloric tiamide 0.1mgL
-1, hydrochloric acid pyroxidine 0.5mgL
-1, nicotinic acid 0.5mgL
-1, inositol 100mg;
Consisting of of above-mentioned startup medium: MS medium, 6-BA 0.2～1.5mgL
-1, NAA 0.08～2mgL
-1, sucrose 25～35gL
-1, agar 6～8gL
-1, add and regulate the pH value before the agar and be pH value 5.8;
Consisting of of above-mentioned inducing culture: MS medium, KT 1.0～5mgL
-1, NAA 0.5～2mgL
-1, sucrose 25～35gL
-1, agar 6～8gL
-1, add and regulate the pH value before the agar and be pH value 5.8;
Consisting of of above-mentioned differential medium: MS medium, IBA 0.2～1.5mgL
-1, 6-BA 2.0～4mgL
-1, sucrose 25～35gL
-1, agar 6～8gL
-1, add that to regulate the pH value before the agar be 5.8;
Above-mentioned condition of culture is: daytime, temperature was 25 ℃, and night, temperature was 18 ℃, intensity of illumination 40umol.m
-2.s
-1, light application time 14hd
-1
(2) select the single colony inoculation of above-mentioned Agrobacterium tumefaciems in containing kanamycin (kanamycin, Kan) in the YEB liquid nutrient medium, 25-34 ℃, under the 160-220rin/m condition, cultivated 18～26 hours, transfer and cultivate in the YEB of no antibiotic medium, bacterium liquid adds an amount of aseptic MS liquid nutrient medium again and is diluted to OD after centrifugal
6000.3～0.7;
Consisting of of above-mentioned YEB medium: every liter contains bacto peptone 5g, yeast extract 1g, beef extract 5g, MgSO
4.7H
2O 0.493g, pH7.0;
(3) above-mentioned callus is cut into small pieces or thin slice, cultivate 1～5 day in advance after, infected Agrobacterium bacterium liquid 4～20 minutes; To adding 15～40mgL in the culture medium (being differential medium) altogether
-1Acetosyringone (acetosyringone AS), cultivates and changes differential medium over to after 3～8 days; Change over to after 10～50 days and select medium to select to cultivate 80～120 days, subculture gets the Kan resistant plant 3～4 times;
Above-mentioned from changing differential medium over to, adding concentration is 400～600mgL
-1Cephalosporin (cefotaxime Cef) suppresses Agrobacterium;
Consisting of of above-mentioned selection medium: differential medium, kanamycin (kanamycin, Kan) 50～100mgL
-1
(4) with the Kan resistant plant of above-mentioned acquisition, obtain transformed plant,, can directly take root as the bud seedling stalwartness of growing thickly of transformed plant through Molecular Detection, thin and delicate as seedling, can change over to and lift medium, after strong sprout, change root media over to;
Above-mentionedly lift consisting of of medium: MS medium, KT 1.0～4.0mgL
-1, 6-BA 0.1～2.0mgL
-1, sucrose 25～35gL
-1, agar 6～8 gL
-1, pH value 5.8～6.0;
Consisting of of above-mentioned root media: MS medium, IBA 1.0～3.0mgL
-1, sucrose 25～35gL
-1, agar 6～8 gL
-1, pH value 5.8～6.0;
(5) after seedling grew 30 days, all can develop into the healthy and strong seedling that takes root more than 90% in root media, after the domestication hardening, plant into the land for growing field crops.
Wherein, described Agrobacterium tumefaciems (Agrobacteriumtumefaciens) the LBA4404 fungus strain that contains binary vector pBin438 of above-mentioned steps (1), its carrier contain betaine aldehyde dehydrogenase gene (BADH) and chimeric NosNpt-II gene and the 35sGUS gene of CaMV35s promotor, mountain spinach.
The prescription of the above-mentioned medium that relates to is:
Start consisting of of medium: MS medium, 6-BA 1.0mgL
-1, NAA 1.0mgL
-1, sucrose 30gL
-1, agar 7.0gL
-1, add and regulate the pH value before the agar and be pH value 5.8;
Consisting of of inducing culture: MS medium, KT 2.5mgL
-1, NAA 1.5mgL
-1, sucrose 30gL
-1, agar 7.0gL
-1, add and regulate the pH value before the agar and be pH value 5.8;
Consisting of of differential medium: MS medium, IBA 1.0mgL
-1, 6-BA 3.5mgL
-1, sucrose 30gL
-1, agar 7.0gL
-1, add and regulate the pH value before the agar and be pH value 5.8;
Consisting of of above-mentioned selection medium: differential medium, kanamycin (kanamycin, Kan) 60mgL
-1
Lift consisting of of medium: MS medium, KT 3.5mgL
-1, 6-BA 1.5mgL
-1, sucrose 30gL
-1, agar 7.0gL
-1, add and regulate the pH value before the agar and be pH value 5.8;
Consisting of of root media: MS medium, IBA 2.0mgL
-1, sucrose 30gL
-1, agar 7.0gL
-1, add and regulate the pH value before the agar and be pH value 5.8.
In the above-mentioned steps (2), bacterium liquid adds an amount of aseptic MS liquid nutrient medium again and is diluted to OD after centrifugal
6000.5.
The callus that relates in the above-mentioned steps (3) infected Agrobacterium bacterium liquid 10 minutes after pre-the cultivation.
The addition of acetosyringone is 30mgL in the above-mentioned steps (3)
-1
The adding concentration of cephalosporin is 500mgL in the above-mentioned steps (3)
-1
In the above-mentioned steps (5), when transplanting tissue cultivating seedling, in the greenhouse, open bottleneck during beginning, reduce humidity gradually, and enhancing illumination, 4～5 days afterwash medium move in the nutritive cube, utilize the intermittent spraying device to keep ambient humidity, through taming hardening after 15-20 days, plant into the land for growing field crops, suitably shade, survival rate can reach more than 95%.
Adopt method of the present invention can within 8～December, obtain to change the plant of BADH gene, set up one repeatably, forest gene transformation technology system that transformation efficiency is high, obtain the transformed plant that salt-resistance improves, and set up supporting tissue-culturing rapid propagation seedling growing process system.
(4) description of drawings
Fig. 1 is the sprouting of giving birth to young sprout then after sterilizing.
Fig. 2 is the callus that is in after infecting in the common cultivation.
(P-is the positive control (1.6kp) of template with the plasmid to Fig. 3 for the PCR of transfer-gen plant detects; 1～4-Kan resistant plant; M-DNA marker; CK-is with the negative contrast of unconverted plant).
Fig. 4 detects (P-is the positive control (1.6kb) of template with the plasmid, and CK-is with the negative contrast of unconverted plant, and 0-does not add the blank of any template DNA, 1～5-transformation plant) for the PCR-Southern of transfer-gen plant.
Fig. 5 is that 5 ‰ NaCl coerce down the grow thickly differentiation of bud of transgenosis (left side) and contrast (right side).
Fig. 6 is that 5 ‰ NaCl coerce down transgenosis (left side) and contrast (right side) seedling growing state.
Fig. 7 is the fast numerous situation of the differentiation of Kan resistance seedling.
Fig. 8 is the differentiation seedling of taking root and cultivating.
Fig. 9 is the transgenosis seedling of transplant survival.
Figure 10 is the transfer-gen plant that has just moved into the land for growing field crops.
Figure 11 is the transgenosis seedling of field-transplanting success.
Figure 12 is the resistance single bud of Kan screening survival after 120 days.
(5) embodiment
The invention will be further described below in conjunction with embodiment:
Embodiment 1:
Vegetable material is the feeding type tetraploid locust improved seeds that Korea S introduces.Explant picks up from the stem segment with axillary bud that young sprout is given birth in the excellent strain in field then, wash with running water, 70% alcohol disinfecting, aseptic water washing 3-5 time is again with mercuric chloride solution sterilization, aseptic water washing 5 times, be seeded in and start in the medium, stem segment with axillary buds begins to sprout after 12 days, sends the tender tip in 20 days, as the induced material of callus.
Wherein, start medium and consist of MS+6-BA 0.3mgL
-1+ NAA 1.5mgL
-1Callus inducing medium is: MS+KT 1.5mgL
-1+ NAA 1.5mgL
-1Differential medium is: MS+IBA 1.0mgL
-1+ 6-BA 3.5mgL
-1The MS medium consists of: KNO
31900mgL
-1, NH
4NO
31650mgL
-1, MgSO
4370mgL
-1, KH
2PO
4170mgL
-1, CaCl
22H
2O 440mgL
-1, MnSO
44H
2O 22.3mgL
-1, ZnSO
47H
2O 8.6mgL
-1, H
3BO
36.2mgL
-1, KI0.83mgL
-1, NaMoO
42H
2O 0.25mgL
-1, CuSO
45H
2O 0.025mgL
-1, CoCl
26H
2O0.025mgL
-1, Na
2-EDTA 37.3mgL
-1, FeSO
47H
2O 27.8mgL
-1, glycine 2.0mgL
-1, hydrochloric tiamide 0.1mgL
-1, hydrochloric acid pyroxidine 0.5mgL
-1, nicotinic acid 0.5mgL
-1, inositol 100mg;
Each medium sucrose concentration is 30gL
-1, agar 6.0gL
-1, pH value 5.8.
Select the single colony inoculation of the Agrobacterium of carrying the BADH gene in the YEB liquid nutrient medium that contains Kan, cultivate 22 hours (25 ℃ 160rin/m), are transferred and cultivate in the YEB of no antibiotic medium, bacterium liquid adds an amount of aseptic MS liquid nutrient medium again and is diluted to OD after centrifugal
6000.4.Callus is cut into small pieces, and pre-incubation time infected Agrobacterium bacterium liquid 4 minutes after 1 day; Train altogether and add 30mgL in the medium
-1AS, cultivate and change differential medium over to after 6 days; Change over to after 30 days and select medium (differential medium+Kan 50mgL
-1), subculture 3-4 time is selected 80 days; From changing differential medium over to, adding concentration is 600mgL
-1Cef suppress Agrobacterium.
The transfer-gen plant that said method is obtained carries out PCR and PCR-Southern detection, and the SDS method is adopted in the extraction of plant genome DNA, the pcr amplification the primer:
1：5′-AGAATGGCGTTCCCAATTCCTGCTC-3′，
2：5′-TTCAAGGAGACTTGATCCATCCCCA-3′；
25 μ l PCR reaction systems: template DNA 2 μ l, 2 kinds of each 0.4 μ mol of primer, each 0.1mmol of dNTP, 0.5U 95 ℃ of pre-sex change of Taq enzyme 10 minutes, extended 1.5 minutes for 1 minute, 72 ℃ in 93.5 ℃ of sex change 1 minute, 58 ℃ of renaturation successively then, circulate 30 times, extended 10 minutes at 72 ℃ at last.Get amplified production 10 μ l and carry out electrophoresis on 1.0% Ago-Gel, GeneGenius full automatic gel imaging analysis system observed result is also taken a picture.In gel electrophoresis, the transformed plant seedling has amplified specific band, consistent with the specific band that positive control amplifies, negative control (unconverted plant) does not amplify any band, the PCR positive detection rate of Kan resistance seedling is about 25%, shows that foreign gene has been incorporated in the genomic DNA of plant.
The callus lines (see figure 5) of the transfer-gen plant that Molecular Detection is obtained and 20 days seedling (about high 2cm) (see figure 6) of differentiation are inoculated into respectively on the differential medium that contains different N aCl concentration (2 ‰, 3 ‰, 4 ‰, 5 ‰, 6 ‰, 7 ‰, 8 ‰), every processing repeats 5 times (bottle), 3 callus of every repetition (bottle) (or resistance seedling), after the cultivation of 20 day time, the NaCl resistance of observing transformed plant improves 2～3 ‰ than unconverted plant.
To obtain (see figure 7) behind the transformed plant differentiation and proliferation, and change over to and lift medium MS+KT 3.5mgL
-1+ 6-BA1.5mgL
-1, after strong sprout, change root media over to.Seedling is at root media MS+IBA 1.0mgL
-1Middle growth all can develop into the healthy and strong seedling (see figure 8) of taking root after 30 days more than 90%.When transplanting tissue cultivating seedling, in the greenhouse, open bottleneck during beginning, reduce humidity gradually, and strengthen illumination, 4～5 days afterwash medium, move in the nutritive cube, note keeping ambient humidity (preferably utilizing the intermittent spraying device), process domestication hardening (see figure 9) after 15-20 days is planted into the land for growing field crops, the (see figure 10) of suitably shading, survival rate reaches more than 95% (sees Figure 11).
Embodiment 2:
Tetraploid locust explant (young sprout stem section) is seeded in startup medium MS+6-BA 1.0mgL after the sterilization
-1+ NAA 1.0mgL
-1In, stem segment with axillary buds begins to sprout after 12 days, sends the tender tip, and the tenderer tip is inoculated into callus inducing medium MS+KT 1.5mgL in 15 days
-1+ NAA 2.0mgL
-1In, the callus of acquisition is transferred to differential medium MS+IBA 1.5mgL again
-1+ 6-BA 2.5mgL
-1In.Each medium sucrose concentration is 25gL
-1, agar 7.0gL
-1, pH value 5.8.
Select the single colony inoculation of the Agrobacterium of carrying the BADH gene in the YEB liquid nutrient medium that contains Kan, overnight incubation (25 ℃ 180rin/m), are transferred and cultivate in the YEB of no antibiotic medium, bacterium liquid adds an amount of aseptic MS liquid nutrient medium again and is diluted to OD after centrifugal
6000.5.Callus is thinly sliced, and pre-incubation time infected Agrobacterium bacterium liquid 10 minutes after 3 days; Train altogether and add 15mgL in the medium
-1AS, cultivate and change differential medium over to after 5 days; Change over to after 30 days and select medium (differential medium+Kan 60mgL
-1), subculture 3-4 time is selected 120 days (seeing Figure 12); From changing differential medium over to, adding concentration is 400mgL
-1Cef suppress Agrobacterium.
The Kan resistant plant that obtains confirms to have obtained transformed plant through Molecular Detection, changes transformation plant over to root media MS+IBA 2.0mgL
-1Middle growth developed into the healthy and strong seedling that takes root after 33 days.Behind acclimatization and transplants, plant into the land for growing field crops, obtained the conversion nursery stock.
Embodiment 3:
Tetraploid locust explant (young sprout stem section) is seeded in startup medium MS+6-BA 1.5mgL after the sterilization
-1+ NAA 0.5mgL
-1In, stem segment with axillary buds begins to sprout after 15 days, sent the tender tip, the tender tip is inoculated into callus inducing medium MS+KT 2.5mgL in 18 days
-1+ NAA 1.5mgL
-1The middle callus that obtains is transferred to differential medium MS+IBA 0.5mgL again
-1+ 6-BA 3.5mgL
-1In.Each medium sucrose concentration is 35gL
-1, agar 8.0gL
-1, pH value 5.8.
Select the single colony inoculation of the Agrobacterium of carrying the BADH gene in the YEB liquid nutrient medium that contains Kan, overnight incubation (30 ℃ 190rin/m), are transferred and cultivate in the YEB of no antibiotic medium, bacterium liquid adds an amount of aseptic MS liquid nutrient medium again and is diluted to OD after centrifugal
6000.6.Callus is thinly sliced, and pre-incubation time infected Agrobacterium bacterium liquid 12 minutes after 5 days; Train altogether and add 35mgL in the medium
-1AS, cultivate and change differential medium over to after 4 days; Change over to after 30 days and select medium (differential medium+Kan 70mgL
-1), subculture 3-4 time is selected 120 days; From changing differential medium over to, adding concentration is 500mgL
-1Cef suppress Agrobacterium.
The Kan resistant plant that obtains confirms to have obtained transformed plant through Molecular Detection, changes transformation plant over to root media MS+IBA 3.0mgL
-1Middle growth developed into the healthy and strong seedling that takes root after 30 days.Behind acclimatization and transplants, plant into the land for growing field crops, obtained the conversion nursery stock.
By above-mentioned 3 examples, detect through PCR and PCR-Southern, this gene transformation method has obtained 70 strain systems that change the BADH gene.
The NaCl resistant proof shows, the grow thickly differentiation of bud of transfer-gen plant is coerced at 6 ‰～7 ‰ NaCl and is not affected down, the normal differentiation substantially also at 8 ‰ o'clock, and unconverted plant promptly is subjected to obvious influence in 5 ‰ differentiation, substantially stopped differentiation at 8 ‰ o'clock, the differentiation of transformed plant improves 3 ‰～4 ‰ to the relevant antagonism of NaCl.3 ‰ NaCl that are grown in of unconverted plant coerce down and the symptom of being injured promptly occurs, and show as amount of growth and reduce, and with the raising of NaCl concentration, the yellow of spray, blade, the withered and phenomena of mortality occur, and amount of growth obviously reduces; And the transfer-gen plant bud of growing thickly is grown in the just appearance symptom of obviously being injured of growth in 6 ‰ o'clock, illustrates that the relevant antagonism of its NaCl has improved 2 ‰～3 ‰.Can transformed plant genetic stability be the important prerequisite of its development and use.This test to first kan select, PCR detects and the plant of salt resistance experiment, after subculture cultivated for 6～8 generations, carried out secondary Kan again and selected (80 days), PCR to detect and the experiment of salt tolerant resistance, the result shows 70% strain system performance genetic stability characteristic (being that Kan selects not occur aetiolation, PCR detects has specific band and salt resistance to improve); The part of 20% the strain system differentiation seedling of growing thickly has aetiolation (doubt and be chimera), is now purifying; 10% strain is obvious yellow, is eliminated.The The above results explanation is eliminated through successive selection, can obtain the transformation plant of inheritance stability fully.
This method set up agriculture bacillus mediated efficiently, forest gene transformation method repeatably, successful is incorporated into betaine aldehyde dehydrogenase gene (BADH) in the tetraploid locust genome, obtained the transformed plant that salt-resistance improves, now successfully be transplanted to the land for growing field crops, obtained a large amount of transformed plants; Tissue-culturing rapid propagation, greenhouse hardening, the nutritive cube of having set up transformed plant transplanted, whole technology of land for growing field crops Cheng Miao.This research is declared national project for bidding " national transgenic plant research and industrialization special project " and is passed, and is now carrying out industrialization development, and the utmost point has utilization and extention to be worth on producing.
Claims (8)
1, a kind of tetraploid locust transgenosis and tissue culture and rapid propagation method, form by following steps:
(1) tetraploid locust is given birth to then the stem section of young sprout, be seeded in after the sterilization and start in the medium, be transferred in the callus inducing medium after 15～30 days, the callus that obtains, after following Agrobacterium tumefaciems (Agrobacterium tumefaciens) the LBA4404 fungus strain transgenosis that contains binary vector pBin438 is handled, inoculate and form the bud of growing thickly in the differential medium;
Consisting of of above-mentioned startup medium: MS medium, 6-BA 0.2～1.5mgL
-1, NAA 0.08～2mgL
-1, sucrose 25～35gL
-1, agar 6～8gL
-1, pH value 5.8;
Consisting of of above-mentioned inducing culture: MS medium, KT 1.0～5mgL
-1, NAA 0.5～2mgL
-1, sucrose 25～35gL
-1, agar 6～8gL
-1, pH value 5.8;
Consisting of of above-mentioned differential medium: MS medium, IBA 0.2～1.5mgL
-1, 6-BA 2.0～4mgL
-1, sucrose 25～35gL
-1, agar 6～8gL
-1, the pH value is 5.8;
Above-mentioned condition of culture is: daytime, temperature was 25 ℃, and night, temperature was 18 ℃, intensity of illumination 40umol.m
-2.s
-1, light application time 14hd
-1
(2) select the single colony inoculation of above-mentioned Agrobacterium tumefaciems in the YEB liquid nutrient medium that contains kanamycin, 25-34 ℃, under the 160-220rin/m condition, cultivated 18～26 hours, transfer and in the YEB of no antibiotic medium, cultivate, bacterium liquid adds an amount of aseptic MS liquid nutrient medium again and is diluted to OD after centrifugal
6000.3～0.7;
Consisting of of above-mentioned YEB medium: every liter contains bacto peptone 5g, yeast extract 1g, beef extract 5g, MgSO
4.7H
2O 0.493g, pH7.0;
(3) above-mentioned callus is cut into small pieces or thin slice, cultivate 1～5 day in advance after, infect Agrobacterium bacterium liquid 4～20min; To being total to culture medium is to add 15～40mgL in the differential medium
-1Acetosyringone (acetosyringone AS), cultivates and changes differential medium over to after 3～8 days; Change over to after 10～50 days and select medium to select to cultivate 80～120 days, subculture gets the Kan resistant plant 3～4 times;
Above-mentioned from changing differential medium over to, adding concentration is 400～600mgL
-1Cephalosporin (cefotaxime Cef) suppresses Agrobacterium;
Consisting of of above-mentioned selection medium: differential medium, kanamycin (kanamycin, Kan) 50～100mgL
-1
(4) with the Kan resistant plant of above-mentioned acquisition, obtain transformed plant,, can directly take root as the bud seedling stalwartness of growing thickly of transformed plant through Molecular Detection, thin and delicate as seedling, can change over to and lift medium, after strong sprout, change root media over to;
Above-mentionedly lift consisting of of medium: MS medium, KT 1.0～4.0mgL
-1, 6-BA 0.1～2.0mgL
-1, sucrose 25～35gL
-1, agar 6～8gL
-1, pH value 5.8～6.0;
Consisting of of above-mentioned root media: MS medium, IBA 1.0～3.0mgL
-1, sucrose 25～35gL
-1, agar 6～8gL
-1, pH value 5.8～6.0;
(5) after seedling grew 30 days, all can develop into the healthy and strong seedling that takes root more than 90% in root media, after the domestication hardening, plant into the land for growing field crops.
2. a kind of tetraploid locust transgenosis as claimed in claim 1 and tissue culture and rapid propagation method, it is characterized in that, described Agrobacterium tumefaciems (Agrobacterium tumefaciens) the LBA4404 fungus strain that contains binary vector pBin438 of step (1), its carrier contain betaine aldehyde dehydrogenase gene (BADH) and chimeric NosNpt-II gene and the 35sGUS gene of CaMV35s promotor, mountain spinach.
3. a kind of tetraploid locust transgenosis as claimed in claim 1 and tissue culture and rapid propagation method is characterized in that the prescription of described medium is:
Start consisting of of medium: MS medium, 6-BA 1.0mgL
-1, NAA 1.0mgL
-1, sucrose 30gL
-1, agar 7.0gL
-1, add and regulate the pH value before the agar and be pH value 5.8;
Consisting of of inducing culture: MS medium, KT 2.5mgL
-1, NAA 1.5mgL
-1, sucrose 30gL
-1, agar 7.0gL
-1, add and regulate the pH value before the agar and be pH value 5.8;
Consisting of of differential medium: MS medium, IBA 1.0mgL
-1, 6-BA 3.5mgL
-1, sucrose 30gL
-1, agar 7.0gL
-1, add and regulate the pH value before the agar and be pH value 5.8; Consisting of of above-mentioned selection medium: differential medium, kanamycin (kanamycin, Kan) 60mgL
-1
Lift consisting of of medium: MS medium, KT 3.5mgL
-1, 6-BA 1.5mgL
-1, sucrose 30gL
-1, agar 7.0gL
-1, add and regulate the pH value before the agar and be pH value 5.8;
Consisting of of root media: MS medium, IBA 2.0mgL
-1, sucrose 30gL
-1, agar 7.0gL
-1, add and regulate the pH value before the agar and be pH value 5.8.
4. a kind of tetraploid locust transgenosis as claimed in claim 1 and tissue culture and rapid propagation method is characterized in that, in the described step (2), bacterium liquid adds an amount of aseptic MS liquid nutrient medium again and is diluted to OD after centrifugal
6000.5.
5. a kind of tetraploid locust transgenosis as claimed in claim 1 and tissue culture and rapid propagation method is characterized in that, the callus that relates in the described step (3) infects Agrobacterium bacterium liquid 10min after pre-the cultivation.
6. a kind of tetraploid locust transgenosis as claimed in claim 1 and tissue culture and rapid propagation method is characterized in that, the addition of acetosyringone is 30mgL in the described step (3)
-1
7. a kind of tetraploid locust transgenosis as claimed in claim 1 and tissue culture and rapid propagation method is characterized in that, the adding concentration of cephalosporin is 500mgL in the described step (3)
-1
8. a kind of tetraploid locust transgenosis as claimed in claim 1 and tissue culture and rapid propagation method is characterized in that, in the described step (5), when transplanting tissue cultivating seedling, in the greenhouse, open bottleneck during beginning, reduce humidity gradually, and enhancing illumination, 4～5 days afterwash medium move in the nutritive cube, utilize the intermittent spraying device to keep ambient humidity, through taming hardening after 15-20 days, plant into the land for growing field crops, suitably shade, survival rate can reach more than 95%.
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Cited By (7)
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| 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 |
| CN101006768B (en) * | 2007-01-24 | 2010-09-08 | 山东省林业科学研究院 | Agrobacterium-mediated Sophora japonica transgene and tissue-culturing rapid propagation method |
| CN101338320B (en) * | 2008-05-22 | 2011-05-25 | 哈尔滨师范大学 | Genetic transformation method of bacopa monnieri mediated by agrobacterium |
| CN102499087A (en) * | 2011-11-01 | 2012-06-20 | 北京林业大学 | Isolated culture and plant regeneration method of silver chain |
| CN102499086A (en) * | 2011-11-01 | 2012-06-20 | 北京林业大学 | Method for breeding locust |
| CN103081805A (en) * | 2013-01-13 | 2013-05-08 | 山西金贝植物科技开发有限责任公司 | Method for efficiently culturing tissues and industrially propagating robinia idaho |
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| JP3144260B2 (en) * | 1995-02-01 | 2001-03-12 | トヨタ自動車株式会社 | Environmental stress-tolerant plant and production method thereof |
| CN1233833C (en) * | 2002-04-05 | 2005-12-28 | 中国科学院植物研究所 | Method for breeding salt-resistant tomato utilizing atriplex hortensis BADH gene transfer |
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| 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 |
| CN101006768B (en) * | 2007-01-24 | 2010-09-08 | 山东省林业科学研究院 | Agrobacterium-mediated Sophora japonica transgene and tissue-culturing rapid propagation method |
| CN101338320B (en) * | 2008-05-22 | 2011-05-25 | 哈尔滨师范大学 | Genetic transformation method of bacopa monnieri mediated by agrobacterium |
| CN102499087A (en) * | 2011-11-01 | 2012-06-20 | 北京林业大学 | Isolated culture and plant regeneration method of silver chain |
| CN102499086A (en) * | 2011-11-01 | 2012-06-20 | 北京林业大学 | Method for breeding locust |
| CN102499086B (en) * | 2011-11-01 | 2012-12-26 | 北京林业大学 | Method for breeding locust |
| CN102499087B (en) * | 2011-11-01 | 2013-02-13 | 北京林业大学 | Isolated culture and plant regeneration method of silver chain |
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| CN103081805B (en) * | 2013-01-13 | 2014-01-29 | 山西金贝植物科技开发有限责任公司 | Method for efficiently culturing tissues and industrially propagating robinia idaho |
| CN106613971A (en) * | 2016-12-08 | 2017-05-10 | 上海杉植物科技有限公司 | Rooting culture method of tissue culture seedlings of fast-growing robinia pseudoacacia Linn.var.inermis DC. |
| CN106613971B (en) * | 2016-12-08 | 2019-08-16 | 上海杉一植物科技有限公司 | A kind of culture of rootage method of the fast-growing without locust tree tissue-cultured seedling |
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