CN1274816C - Molecular breeding method for quickly obtaining great amount of new transgenic plant varieties - Google Patents

Abstract

The present invention provides a method for rapidly obtaining a large amount of transgenic plant novel species. Resistance callus obtained after agricultural bacilus conversion is divided into regeneration seedlings by a somatic embryogenesis path according to the method, so the regeneration efficiency of transgenic seedlings is greatly improved. Goal resistance plants are filtered by adding a goal resistant filtering reagent in the somatic embryo germination and growth process, so the obtaining rate of goal resistance transgenic seedlings can be greatly enhanced, and the operation capacity of detection at a later stage is reduced. A great amount of goal resistance transgenic alfalfa regeneration seedlings can be obtained by using the technology for 12 weeks, the requirements for biological detection in a large scale and good plant filtration at present are satisfied, field experiments are combined, and a large amount of good species with a good phenotype can be obtained. The technology system has obvious economical and social benefits during forage grass breeding.

Description

The molecular breeding method of a large amount of transgenic plant new variety of a kind of quick acquisition

Technical field

The target resistance screening three who the present invention relates to utilize agriculture bacillus mediated genetic transformation, intends transforming the high-frequency plant regeneration system of plant and regeneration plant combines and cultivates the method for new variety (being), belongs to biological technical field.

Background technology

Plant genetic transform (genetic transformation) be the extraneous nucleotide fragment change plant tissue over to, cell makes foreign DNA can keep, express, also can pass to offspring's technology.It is to be accompanied by molecular biology and plant cell tissue's culture technique constantly develops, a new technology of just rising at the mid-1970s, develop over nearly 20 years very rapid.From 1985, people such as Adang have reported that the B.t gene successfully imports tobacco, and, foreign gene is expressed (Gene in the transgene tobacco base, 1985,36:289～300.), transgenic plant have expanded to cash crop, food crop, vegetables, flowers, medicinal plant, fruit, trees and herbage etc. at 35 interior genus, more than 120 species now.Now, some have all realized commercialization through crop new variety such as tomato, rape, tobacco, summer squash, flax, corn, soybean and the cotton of genetically engineered improvement (or cultivation), the proterties that relates to comprises (kingdom's English such as improvement of quality-improving, pest-resistant, antiweed, disease resistance, 1998, science, 50,5:38～40).But biotechnology breeding is but made slow progress on some species, and its major cause is not normally set up the technical system of the efficient genetic transformation of suitable relevant species, and the plant regeneration system of this technical system medium-high frequency is the key that realizes genetic improvement.

By genetic engineering technique clover being carried out genetic improvement is the main application fields of biotechnology in the herbage molecular breeding in recent years.Its major cause is, clover have the title of " king of herbage ", is various countries' first-selected grass seeds of developing animal husbandry in this world.In addition, alfalfa content height is the good plant material as functional proteins such as plant bioreactor production vaccines.As far back as 1986, MariaDeak (Plant Cell Reports, 5:97～100) just utilized agrobacterium-mediated transformation that NPT-gene is imported the tender stem segments of clover, and obtains transfer-gen plant, has realized the genetic transformation to clover.1988, people such as Mireille (Plant Cell Reports, 7:512～516) imported alfalfa blade recipient cell with kalamycin resistance gene with the Agrobacterium infestation method.Afterwards, also more by multiple method for transformation to the report of the research of the genetic transformation of clover, also obtained many achievements.Halluin (CropSci, 1990,30:866～871) transforms clover by Agrobacterium with anti-herbicide gene and succeeds, and people such as Hill (Bio/Technology, 1991,9:373～377) utilize Agrobacterium that the Bt toxoprotein gene is led the clover achieving success.2000, China Lv Deyang (Acta Genetica Sinica, 27,4:331～337; Chinese science, 4,8:342～348) reported domestic the first transgenic alfalfa Study on Regeneration, but only obtain 11 strain regenerated transgenic seedlings.This is to the efficient genetic transformation of clover and to improve be not enough.Take a broad view of the application of domestic and international biotechnology on the clover genetic improvement, transforming back regrowth acquisition efficient low is the subject matter of its development of restriction.The kanamycin-resistant callus tissue majority is within 40%, and the differentiation rate of kanamycin-resistant callus tissue also only is 1～5%, and this has had a strong impact on the development of clover biotechnology breeding.Therefore, it is necessary setting up complete clover biotechnology molecular breeding system.

The present invention is exactly at disadvantages of background technology and defective, solved the difficult problem of clover regeneration, perfect regeneration system and the genetic conversion system of clover increased substantially the pick-up rate of the transgenic regenerated plant of clover, for the molecular breeding of clover has been established solid basis.In addition, by specific experimental design, make that the pick-up rate of kanamycin-resistant callus tissue reaches more than 50% behind the clover genetic transformation, the incidence of the somatic embryo of kanamycin-resistant callus tissue reaches more than 85%, the positive rate of resistance regrowth reaches more than 80%, and directly obtains to have the regrowth system of target gene resistance.By in the genetic transformation process, carrying out the selection of elite plant strain, and expand numerously, accelerated the acquisition of good transgenosis new variety.Have certain social benefit and economic benefit, can be applicable to the herbage improvement of China.

Advantage of the present invention:

(1) the time weak point is simple to operate, can carry out genetic improvement to clover easily.

(2) transformation efficiency height, the resistance regrowth is many, can provide a large amount of individual confessions of transgenosis to select subsequently.

(3) solved the quick breeding problem of the good plant of special proterties, it is seedling that the strain of a large amount of good character can be provided fast, needn't obtain seed by traditional planting patterns and just can carry out improved seeds (being) selection, has quickened breeding process.

Summary of the invention

The object of the present invention is to provide a kind of method of clover being carried out genetic improvement by the biotechnology measure, thereby realize the high-performance bio technology breeding of clover, so that improve the resistance of clover from many aspects and improve quality, and for utilizing clover to do that bio-reactor is produced vaccine or other functional protein provides technical support.The key technical indexes is: the pick-up rate of kanamycin-resistant callus tissue is more than 50% behind the clover genetic transformation, and the differentiation rate of the kanamycin-resistant callus tissue that the screening back obtains is more than 85%, and the regrowth acquisition time is about 10 weeks, and wherein the transgenic seedling pick-up rate is more than 80%.Its main contents comprise: be suitable for clover regenerated minimum medium, the proportioning of inducing and keep substratum of callus, somatic embryo inducement and sprouting, the acquisition of Agrobacterium-mediated Transformation and resistant calli, resistant calli is somatic induces, the screening and the acquisition of the sprouting of resistance somatic embryo and transgenosis resistance regrowth, the Molecular Detection of resistance regrowth and expanding propagation, cell simulations resistant proof etc.

In the present invention, term " target gene " presentation code specific protein instructs the gene of plant biology characteristic, as genes such as the drought resisting of plant, pest-resistant, waterlogging-resistant, pattern, height.

Term " is selected pressure " and is illustrated in the screening that will screen medicament by finite concentration in the process that obtains regenerated transgenic seedling, and the concentration of screening medicament is selected to press exactly, and the high low reaction of the concentration of medicament is selected the height of pressure.

Term " marker gene " expression refers in the transgenosis process, is the transgenic plant that obtain the target gene mark property, is used for the gene of assisted Selection to be called marker gene.Marker gene is a resistant gene often, and the cell, tissue, organ, plant materials that have marker gene are to the particular agent strong resistance.

The gene that small-molecule substance that osmoregulation works in plant materials that term " resistance gene " presentation code is specific or coding impel the cell membrane stability material, its function are that plant has stronger tolerance to environment stress.The resistance gene comprises the gene that the drought resistance that makes plant, frost resistance, waterlogging-resistant property, insect-resistance etc. improve.As the HVA1 gene, the drought resistance of plant is improved, belong to anti-drought gene, the rstB gene can make the salt tolerance of plant improve, and belongs to salt resistant gene.Other gene relevant with the resistance of plant of finding also has trimethyl-glycine synthase gene, sorbyl alcohol synthase gene, SOD family gene, SOS family gene etc. at present.

The side of a large amount of transgenic plant new variety of a kind of quick acquisition that the present invention proposes is the example explanation with the alfalfa clover.Obtain the molecular breeding method of a large amount of transgenic plant new variety of alfalfa and efficient genetic transformation thereof fast, comprise following step:

(1) clover explant preparation: the fresh and tender material of alfalfa (cotyledon, hypocotyl, blade, petiole, root etc.) of choosing the good biological character of tool is an explant.Explant such as cotyledon, hypocotyl can obtain by aseptic seedling.The full seed of choosing good alfalfa kind soaks 2min in 75% alcohol, with the 10min that sterilizes in 0.1% the mercuric chloride solution, use aseptic water washing 3-5 time more then, and being inoculated in germinates on the 1/2MS substratum obtains aseptic seedling.Be that desirable cotyledon, hypocotyl etc. are done explant after one week.

(2) pre-cultivate and Agrobacterium is infected: after aseptic seedling forms, get cotyledon and laterally be cut into wide little of 1mm, hypocotyl is cut into the segment that 2-4mm grows, and the SH substratum MSH that is inoculated into improvement is (with SH (Sckenk ﹠amp; Hilderebrantdt 1972 Can J Bot, 50:199-204) macroelement+MS (Murashige ﹠amp; Skoog medium, 1962, Physiol.Plant 15:473-497) trace element and molysite+caseinhydrolysate 1～2mg/L+VB ₁9.9mg/L+VB ₆9.5mg/L+ nicotinic acid 4.5mg/L+ agar 8g/L+30g/L sucrose is the substratum of basal component, pH5.8, below identical) additional 2.0mg/L 2, (kinetin KT) cultivates 1～2 day in advance for 4-D and 0.025mg/L kinetin.(agrobacterium strains is LBA4404 will to have the Agrobacterium bacterium liquid of target gene, purchase in ancient cooking vessel state biotech firm), target gene is connected in eukaryotic expression plasmid 3301 (ppt resistance), on plasmid 1301 (hygromycin resistance) and the plasmid pBI121 (kalamycin resistance) (above plasmid is all purchased the biotech firm in clontech), with the additional 2.0mg/L 2 of MSH, (do not add agar solidifies the liquid nutrient medium of 4-D and 0.025mg/L KT, pH5.2～6.0) be diluted to OD value 0.3～0.6 after, the pre-incubated clover explant (cotyledon of process, hypocotyl etc.) be immersed in the Agrobacterium bacterium liquid and soaked 5 minutes, then, material is pulled out from bacterium liquid, be placed on the aseptic filter paper and blot, (MSH adds 2.0mg/L 2 to forward the callus of induce substratum to, the solid medium of 4-D and 0.025mg/L KT solidifies with agar 8g/L) on cultivate altogether and began to show to the Agrobacterium spot in 4～7 days.

(3) screening of clover resistant calli and degerming: explant and Agrobacterium have begun to form callus after through 4～7 days common cultivation.At this moment, all explants and callus transferred to contain the callus of induce that screens medicament and keep substratum that (MSH adds 2.0mg/L2, the solid medium of 4-D and 0.025mg/L KT solidifies with agar 8g/L) on carry out kanamycin-resistant callus tissue screening and degerming (Fig. 1).The degerming medicament can be with carboxylic Bian penicillin or the thiophene saitomycin of 1500～300mg/L or the saitomycin of 200～400mg/L.The consumption of screening with medicament (select to press, as: PPT, kantlex, Totomycin etc.) was changed a subculture in per 10～14 days gradually from low to high.With Totomycin (hpt) serves as select to press (corresponding to 1301 carriers) to begin to screen for two weeks with 10mg/L, after forward to and screen on the substratum that contains 20mg/L hpt 2～3 times, promptly finish the kanamycin-resistant callus tissue screening process; With weedicide ppt (phosphinothricin, commodity are called Basta, when effective constituent glufosinate) pressing (corresponding to 3301 carriers) for selection, screened for two weeks with 5mg/L ppt during beginning, after forward 10mg/L ppt to and screened for two weeks, at last, screen the screening of promptly finishing kanamycin-resistant callus tissue two weeks with 15mg/L ppt.Make when select pressing (corresponding to the pBI121 carrier) of kantlex (kam), screening process is to screen for two weeks with 50mg/L kam earlier, after transfer on the screening culture medium of 100mg/L kam and screened for 2～4 weeks, finish the screening of kanamycin-resistant callus tissue.Certainly, according to the difference of marker gene, also have other screening medicament available (as: tsiklomitsin, paraxin, spectinomycin etc.), but total method is to select to press from low to high.Eliminate the callus of brownization and water stainization when noting each subculture.

(4) alfalfa callus somatic embryo inducement: in clover resistant calli screening process, the part callus can change oyster gradually into, promptly reach the state (claiming embryo callus subculture usually) before somatic embryo takes place, forward the callus of this oyster to division culture medium MSH additional 0.3～0.6mg/L KT, carry out somatic embryo inducement (Fig. 2) on the antibiotic substratum of degerming of lower concentration resistant calli screening medicament and lower concentration.As: as if being the degerming medicament with the Pyocianil, the interpolation concentration of this moment can drop to 150mg/L by 200 original～400mg/L; With PPT is that kanamycin-resistant callus tissue is selected to press, and the selection of this moment is pressed concentration to select to press 15mg/L to drop to about 2～5mg/L by high density and got final product.On the somatic embryo inducement substratum, can there be a large amount of torpedo-shaped embryoids to form in general about 20 days.

(5) acquisition of the sprouting of somatic embryo of clover and resistance seedling: the torpedo-shaped embryoid that is taken at the anti-screening medicament that forms on the somatic embryo inducement substratum, be transferred to the additional resistant calli screening of 1/2MS medicament (as: PPT 2～5mg/L, kantlex 25～50mg/L or Totomycin 5～10mg/L; The screening medicament selection change because of carrier) and the pairing objective trait of the target gene that is transformed (be mainly abiotic stress proterties such as anti-salt, drought resisting; Gene that this experiment tries is salt resistant gene rstB, so be screening reagent with NaCl, selected concentration is 0.5%; If the gene that tries is anti-drought gene, available PEG comes assisting sifting) the substratum of screening reagent on make embryoid sprouting (Fig. 3,4).What note is when carrying out resistance screening, determine the initial resistance to the objective trait screening medicament of intending screening (determine the non-transgenic seedling is answered reagent to the selection markers gene pairs and to the resistance of the corresponding degeneration-resistant proterties of target gene) of non-transgenic seedling.Usually used screening method is screening indirectly, promptly utilizes the pairing proterties of selection markers gene to screen, as bar gene (having Herbicid resistant), Kam ^rGene (having kalamycin resistance) is commonly used to the gene that makes marks, and only screens transfer-gen plant with PPT or kantlex during screening.The method of marker gene assisting sifting is because some uncertain reasons make the false positive rate of resistance seedling higher.The present invention proposes with selection markers gene and target gene in conjunction with dual method for screening target resistant transgenic seedling pick-up rate to be increased substantially.With obtaining a large amount of resistance regrowths (Fig. 5) after 0.5% NaCL and the 2mg/L PPT screening, the pick-up rate of target resistance (anti-salt) regenerated transgenic seedling is more than 80% in this experiment.

(6) Molecular Detection of resistant plant: the resistance seedling that obtains is carried out total DNA extraction respectively according to the SDS method; the PCR that carries out target gene then detects; Southern detects; Northern detects; immunoassay etc., respectively the target gene that transforms in different level detection in the genomic integration situation of clover, transcribe situation and expression level etc.The PCR of antagonism regrowth detects (Fig. 7,8) in this experiment, and Southern detects (Fig. 9) and finds that the positive plant rate reaches more than 80%.

(7) expanding propagation of resistant transgenic regeneration plant: the present invention's special resistance seedling in having solved the experiment of common genetic transformation is few and can not be in the contradiction of subsequent experimental such as carry out that biological assay, resistance are determined in the present age, can obtain a large amount of special resistance regrowths of transgenosis in the present age/year, can carry out the biology of special resistant transgenic seedling the present age/year measures, shorten the time that new variety are selected, accelerated the paces of molecular biosciences breeding.

The breeding of the transgenosis of special resistance regeneration clover can be adopted cottage propagation on substratum, also can get the young tender material evoked callus such as blade, petiole of regrowth, carries out expanding propagation in processes such as carrying out somatic embryo inducement and sprouting.Comparatively speaking, the breeding coefficient of preceding a kind of modes of reproduction is lower, and two months cottage propagations of a strain Cheng Miaojing can obtain 6～10 seedlings for twice, and then a kind of method can obtain hundreds of to several thousand seedlings in two months.The operating process of preceding a kind of method is: the stem section that aseptic clover resistance regrowth is cut into 3～4cm with scissors, go up requirement for every section and have two stipes at least, then,, to the 1/2MS substratum, the new regenerated root of formation will be arranged in general about 20 days and become whole plant by its growth polarity cuttage.The operating process of a kind of method in back is: blade, the handle of getting the resistance regrowth cuts or the stem section is done explant, be cut into the segment of 3 millimeters sizes, place the MSH substratum to add 2.0mg/L 2, on the solid medium of 4-D and 0.025～0.05mg/L KT, after general 4 days, callus can form, and promptly has a large amount of somatic embryos to take place time 20 days.At this moment, the callus that will have somatic embryo forwards division culture medium (the MSH substratum adds 0.4mg/L KT) to and goes up or the 1/2MS substratum, and somatocyte promptly can be sprouted after 30 days becomes complete regrowth.Finishing the whole regeneration period only must 50～60 day.

(8) transplanting of resistance regeneration plant and biology are measured: the resistance clover regrowth of acquisition promptly can be transplanted when root reaches more than the 5cm.Open earlier before the transplanting and cultivate bottleneck exercise 3～5 days, at room temperature transplanting survival rate reaches 85%.In humidity is 80%, and temperature is controlled at 25 ℃ daytime, is controlled at 20 ℃ evening, and surviving rate can reach more than 95% in the growth cabinet of photoperiod 14h illumination/10h dark.

After expanding propagation, intend improveing the sub-district test of target resistance through the clover regrowth that obtains after the Molecular Detection with target gene integration.Testing location is chosen in can be provided under the physical environment or artificial environment of necessarily coercing injury.During this time, write down the not growth of homophyletic system and report situations, and then determine that good strain system, separation obtain the new lines that has the target resistance and grow fine.

Used substratum in this experiment all is 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

The screening of Fig. 1 clover kanamycin-resistant callus tissue.

Fig. 2 alfalfa callus somatic embryo inducement.

Fig. 3 somatic embryo of clover is sprouted.

Fig. 4 resistant transgenic clover regrowth.

The NaCL of Fig. 5 anti-0.5% and the transgenic alfalfa plants of 2mg/L PPT.

The transplantation of seedlings of Fig. 6 transgenic alfalfa resistance survives.

Fig. 7 transforms resistance clover seedling target gene (rstB) the PCR detection that the back obtains, and M is Mark, and ck+ is target gene PCR result, and ck-is non-transgenic plant PCR result, and 1,2,5,6,7,9 for the PCR detection is transfer-gen plant, and 3,4,8 is the non-transgenic plant.

Fig. 8 resistance clover regrowth selection markers gene (bar gene) PCR detects,, M is Mark, ck+ is target gene PCR result, ck-is non-transgenic plant PCR result, 1,2,5,6,7,9 transfer-gen plants that detect target gene (rstB) for PCR also detect mark (Bar) gene.

Fig. 9 transgenic alfalfa regrowth target gene (salt-resistant related gene rstB) Southern is hybridized detection, M is Mark, and ck+ is a target gene PCR product results of hybridization, and ck-is a non-transgenic plant results of hybridization, 1-7 is the transfer-gen plant results of hybridization, and 8 is unconverted successful plant.

Embodiment

Implement reagent

The inorganic 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 limited liability company product.Above reagent is available from provisioning section of China Agricultural University (other reagent company of market is also on sale).

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.

Embodiment 1

Experimental cultivar is the Peru clover, purchases in academy of agricultural sciences's herding institute.Be inoculated on the 1/2MS substratum after getting Peru's alfalfa seed sterilization, get cotyledon and hypocotyl after the week and place callus induction and keep substratum (composition is the additional 2.0mg/L 2 of MSH, the solid medium of 4-D and 0.025mg/L KT) to go up pre-the cultivation 24 hours.Subsequently at the additional 2.0mg/L 2 of MSH substratum, 5～10 times of middle immersions 5 minutes of Agrobacterium (LBA4404) bacterium liquid (shaking bacteria concentration is OD value 0.8～1.0) that have salt resistant gene (rstB) of the liquid nutrient medium dilution of 4-D and 0.025mg/L KT, on aseptic filter paper, blot bacterium liquid and be placed on the additional 2.0mg/L 2 of MSH substratum, cultivated altogether 5 days on the solid medium of 4-D and 0.025mg/L KT (callus of induce and maintenance substratum).Then, forwarding the callus of induce that contains 5mg/L ppt and 250mg/L carboxylic Bian penicillin to screened for two weeks on the substratum with keeping.Afterwards, the resistant calli that will screen acquisition again forwards the callus of induce that contains 10mg/L ppt to and keeps two weeks of screening on the substratum, screens once with 15mg/L ppt at last, finishes the screening of kanamycin-resistant callus tissue.The kanamycin-resistant callus tissue that screening is obtained forwards additional 3mg/L ppt to, (MSH adds 0.3 respectively to the division culture medium of 150mg/L carboxylic Bian penicillin, 0.4,0.5,0.6mg/L KT, sucrose concentration are 15g/L, 20g/L, amount to 8 combinations) but go up all that the inductor somatic embryo forms, after 20 days the somatic embryo of inducing formation forwarded on the no hormone culture-medium 1/2MS of additional 2mg/L PPT of 1/2MS and 0.5% NaCL and make its sprouting and screen anti-salt plant.Strain resistance regrowth surplus this experiment obtains 600.The resistance seedling that screening obtains is found that through PCR detection and Southern hybridization detection 82% resistance seedling all has target gene.Selection growth conditions strain fine individual plant preferably carries out expanding propagation, has obtained nearly 100 target resistances elite plant strain (each at least 500 seedling of strain system) preferably then.Improved the pick-up rate of positive seedling with this method greatly than traditional method, and, just can obtain elite plant strain then.

Embodiment 2:

Experimental cultivar is Baoding clover, purchases in academy of agricultural sciences's herding institute.Other step and minimum medium are with embodiment 1, used screening medicament is kantlex (purchasing yuxin through biotech firm of section), initial selected is pressed and is 50mg/L, screening time was two weeks, after the 100mg/L kantlex screened for 3 weeks, the resistant calli that obtains is forwarded to contain the 25mg/L kantlex, (MSH additional 0.4 for the division culture medium of 150mg/L carboxylic Bian penicillin, 0.5mg/L kinetin, sucrose concentration are 20g/L, two combinations) upward inductor somatic embryo differentiation.The back is containing the 25mg/L kantlex, the anti-salt plant of screening on the 1/2MS substratum of NaCl 3g/L, and this experiment obtains 321 resistance regrowths, detects through PCR detection and Southern hybridization, finds that 85% resistance seedling all has target gene.The tissue culture expanding propagation is carried out in the excellent preferably strain of growth conditions wherein, obtain nearly 100 elite plant strains (each at least 500 seedling of strain system) then, and nearly 90% all blossom and bear fruit.This method has improved the efficient of breeding of the pick-up rate of excellent strain and elite plant strain greatly than ordinary method.

Embodiment 3:

Experimental cultivar is No., middle lucerne, purchases in academy of agricultural sciences's herding institute.Other step and minimum medium are with embodiment 1, used screening medicament is Totomycin (purchasing yuxin through biotech firm of section), initial selected is pressed and is 10mg/L, screening time was two weeks, after 3 weeks of 20mg/L hygromycin selection, the resistant calli that obtains is forwarded to contain the 3mg/L Totomycin, (MSH additional 0.3 for the division culture medium of 150mg/L carboxylic Bian penicillin, 0.4mg/L kinetin, sucrose concentration are 20g/L, two combinations) upward inductor somatic embryo differentiation.Then, the anti-salt plant of screening on the 1/2MS substratum that contains Totomycin 3mg/L and NaCL 3g/L, this experiment obtains 452 anti-salt regrowths, detects and Southern hybridization detection through PCR, finds that 81% resistance seedling all has target gene.The tissue culture expanding propagation is carried out in excellent preferably strain to growth conditions, obtains nearly 100 elite plant strains (each at least 500 seedling of strain system) then, and 90% seedling all blossoms and bears fruit.This method has improved the efficient of breeding of the pick-up rate of excellent strain and elite plant strain greatly than conventional breeding method.

To sum up, mainly select effect better when the resistance gene pairs plant genetic transformation about the molecular breeding method of a large amount of transgenic plant new variety of quick acquisition/be.The key of this technology is: (1) sets up the high frequency regenerating system of this kind of plant; (2) regeneration plant behind the commentaries on classics target resistant gene improves a lot than the resistance of non-transgenic plant; (3) the dual screening resistant plant of marker gene and target gene; (4) Yan Ge Molecular Detection determines that target gene transforms successfully; (5) the excellent strain that does very well of a large amount of breedings, in the strain system of breeding, carry out new lines/kind seed selection.

Claims (15)

1. a molecular breeding method that obtains a large amount of transgenic plant new variety fast comprises (1) preparation explant; (2) described explant is containing 2.0mg/L 2, after cultivating in advance on the MSH substratum of 4-D and 0.025mg/L kinetin, infect explant with the Agrobacterium bacterium liquid that contains target gene, the basal component of wherein said MSH substratum comprises the SH macroelement, MS trace element and molysite, the 1-2mg/L caseinhydrolysate, 9.9mg/L VB ₁, 9.5mg/L VB ₆, 4.5mg/L nicotinic acid, 8g/L agar and 30g/L sucrose; (3) containing on the described substratum that selection that the degerming drug concentration raises gradually presses the screening resistant calli and removing Agrobacterium; (4) evoked callus somatic embryo on the described substratum that contains 0.3-0.6mg/L kinetin and lower concentration selection pressure; (5) somatic embryo is sprouted, and utilizes marker gene and the pairing medicament of target gene to screen the resistant transgenic regrowth; (6) the resistance seedling that transforms the back acquisition is carried out Molecular Detection; (7) expanding propagation resistant transgenic regrowth; (8) the antagonism regenerated transgenic seedling carries out biological assay, obtains excellent strain, and wherein said plant is a clover.

2. the process of claim 1 wherein that described target gene is the resistance gene.

3. the method for claim 2, wherein said resistance gene is anti-drought gene HVA1 or resistant gene of salt rstB.

4. the process of claim 1 wherein that described degerming medicament is to suppress the Agrobacterium growth or make its dead medicament.

5. the method for claim 4, wherein said degerming medicament is a Pyocianil, thiophene saitomycin or cephalo are mould.

6. the process of claim 1 wherein described selection press be described marker gene the medicament of corresponding resistance.

7. the method for claim 6, wherein said medicament is a Totomycin, kantlex, ppt or tsiklomitsin.

8. the method for claim 7, it is characterized in that the concentration of described Totomycin in screening resistant calli process by the 10mg/L of lower concentration through carrying out the transition to the higher 20mg/L of concentration 2 weeks, concentration is 3mg/L in the process of inductor somatic embryo.

9. the method for claim 7, through carrying out the transition to the higher 100mg/L of concentration 2 weeks, concentration is 25mg/L to the concentration of wherein said kantlex in screening resistant calli process in the process of inductor somatic embryo by the 50mg/L of lower concentration.

10. the method for claim 7, the concentration of wherein said ppt in screening resistant calli process is by the 5mg/L of lower concentration, through carrying out the transition to the medium 10mg/L of concentration 2 weeks, through carrying out the transition to the higher 15mg/L of concentration 2 weeks, concentration is 2-5mg/L in the process of inductor somatic embryo again.

11. the method for claim 1 is characterized in that being divided in the process of somatic embryo at evoked callus, and the sucrose concentration in the described substratum is reduced to 15-20g/L.

12. the method for claim 1, the differentiation that it is characterized in that the resistant calli that obtains through resistance screening is through the somatic embryo inducement approach, and the working concentration of kinetin is 0.3～0.6mg/L.

13. the method for claim 1 is characterized in that somatic embryo after inducing formation, utilizes marker gene and the dual screening of target gene in its sprouting becomes the process of resistance clover transgenic seedling.

14. the method for claim 1 is characterized in that behind the resistant transgenic regrowth that Molecular Detection obtains, with its through callus induce and expanding propagation is carried out in differentiation.

15. the method for claim 1 is characterized in that the resistant transgenic regrowth via expanding propagation, screening acquisition excellent strain/kind.

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