CN101935672B - Agrobacterium rhizogenes transformation method for living body growth cone of woody plant - Google Patents
Agrobacterium rhizogenes transformation method for living body growth cone of woody plant Download PDFInfo
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Abstract
The invention discloses an agrobacterium rhizogenes transformation method for a living body growth cone of a woody plant, which comprises multiple steps of preparing agrobacterium rhizogenes engineering bacterium osmosis transformation liquid, converting and inoculating the living body growth cone, coculturing the growth cone and agrobacterium rhizogenes engineering bacteria, screening resistant sprouts, culturing resistant branches, identifying a transgenic plant, and the like. The invention breaks through two technical bottlenecks of difficult gene transformation and difficult regeneration of the woody plant, is independent of plant genotype and tissue culture, has the advantages of high transformation efficiency, simple operation, low cost, and the like and is suitable for most of woody plants, such as tea trees, and the like, especially the woody plant with full terminal buds.
Description
Technical field
The present invention relates to a kind of plant gene method for transformation, particularly the live body growing tip Agrobacterium-mediated Transformation method of xylophyta such as tea tree.
Background technology
Plant genetic transforms and is meant through certain technology or approach; Foreign gene is imported in the recipient plant genome; Make its genetic stability in recipient plant, and give plant new economical character, noticeable change of, high yield anti-, high-quality, metabolic substd content or the like like height.
During plant genetic transformed, genetic recipient had two types of vitro culture material and living materials usually.Utilize vitro culture material such as protoplastis, suspended culture cell, callus, sporule and originally explant etc. promptly realize genetic transformation as genetic recipient through tissue culture and plant regeneration, be present topmost method.In addition, use living materials such as whole plant, seed or pollen etc., directly foreign gene is imported recipient plant and also obtained success as genetic recipient.
The plant genetic transformation technology can be divided into two big types: one type is the direct gene transfer techniques, comprises that particle bombardment, protoplasm body, liposome method, pollen tube passage method, electricity swash conversion method, polyoxyethylene glycol conversion method etc., and wherein particle bombardment is representative.Another kind of is the method for transformation of biological mediation, mainly contains two kinds of Agrobacterium-mediated Transformation method and viral conversion methods, and wherein the Agrobacterium-mediated Transformation method is easy and simple to handle, cost is low, transformation efficiency is high, is widely used in dicotyledons even monocotyledonous genetic transformation.
The genetic transformation of xylophyta is a great problem up to now.As everyone knows, xylophyta is unusual difficulty through callus regeneration in tissue culture, and this is the restriction xylophyta carries out genetic transformation through tissue culture and plant regeneration primary factor.In addition; Xylophyta generally contains a large amount of polyphenol; Polyphenol is a large amount of rapidly accumulation at wound cell place, are a kind of self-protective mechanisms for plant, if but the polyphenol at wound cell place is overweight then inhibited to agriculture bacillus mediated gene transformation; Even Agrobacterium itself had lethal effect, thereby cause the Agrobacterium-mediated Transformation method to show in the genetic transformation of xylophyta that transformation frequency is very low, effect is unstable, be difficult to problems such as recurrence based on tissue culture and plant regeneration.Therefore, setting about the optimization and the innovation of xylophyta genetic transformation technology, to advancing the development of xylophyta genetically engineered significant, also is simultaneously to utilize pressing for that modern molecular breeding technology cultivates improved seeds.
Summary of the invention
In view of this, the object of the present invention is to provide the live body growing tip Agrobacterium-mediated Transformation method of a kind of xylophyta, broken through xylophyta gene transformation difficulty and regeneration difficulty two big technical bottlenecks; Do not rely on plant gene type and tissue culture; Have the transformation efficiency height, simple to operate, advantage such as with low cost; Be applicable to most of xylophytas such as tea tree, especially the full xylophyta of terminal bud.
For achieving the above object, the live body growing tip Agrobacterium-mediated Transformation method of xylophyta of the present invention may further comprise the steps:
The preparation of a, Agrobacterium engineering bacteria infiltration conversion fluid: the Agrobacterium engineering bacteria that will contain goal gene is inoculated in the liquid nutrient medium that contains the screening pressure and cultivates, as bacterium liquid OD
600Value reaches at 0.5～1.0 o'clock, in bacterium liquid, adds screening and presses, and add permeate agent and tensio-active agent, is mixed with Agrobacterium engineering bacteria infiltration conversion fluid;
The conversion of b, live body growing tip inoculation: in leaf layer medial temperature is that 18～26 ℃, medial humidity are under 70～80% the condition, and Agrobacterium engineering bacteria infiltration conversion fluid is injected on the growing tip in the xylophyta young sprout terminal bud bud bud;
The common cultivation of c, growing tip and Agrobacterium engineering bacteria: the young sprout terminal bud after will transforming cover the suction material which can retain moisture and the condition of the cooling moisture-retaining that shelters from heat or light with the sunshade net under cultivated altogether 2～10 days, control leaf layer medial temperature is that 18～26 ℃, medial humidity are 70～80%;
The screening of d, resistant buds: after cultivating end altogether, on the growing tip of young sprout terminal bud, add screening and press, suppress the division of non-transformed cell in the growing tip, make the transformant proper splitting and be divided into resistant buds;
The cultivation of e, resistance branch: carry out field fertilization and insect pest preventing and controlling management by ordinary method, in time cut the branch that unconverted is handled, make resistant buds grow into the resistance branch;
The evaluation of f, transfer-gen plant: the antagonism branch carries out transgenic and identifies that the plant that the result is positive is transfer-gen plant.
Further, the terminal bud of said xylophyta is full;
Further, said xylophyta is a tea tree;
Further, said step a is inoculated in the Agrobacterium engineering bacteria that contains goal gene to contain in the LB liquid nutrient medium that screens pressure, Streptomycin sulphate and Rifampin to cultivate, as bacterium liquid OD
600Value reaches at 0.6～0.8 o'clock; In bacterium liquid, adding screening presses; And to add concentration be that sucrose, the concentration expressed in percentage by volume of 100g/L is that 0.02～0.05% Silwet L-77 and concentration expressed in percentage by volume are 0.01～0.05% tween, is mixed with Agrobacterium engineering bacteria infiltration conversion fluid;
Further, said step b transforms inoculation full period at xylophyta young sprout terminal bud, and does not damage when transforming inoculation or minor injury's growing tip only;
Further; Said step c covers the young sprout terminal bud after transforming with the strong cotton of suction moisture retention earlier; On cotton, cover 50%～90% sunshade net again; Growing tip and Agrobacterium engineering bacteria were cultivated 6 days altogether, squirted cotton with aqueous sucrose solution in wherein preceding 2 days to preserve moisture, back 4 days waters squirt cotton and preserve moisture.
Beneficial effect of the present invention is: in view of 1. Agrobacterium to the wound cell or do not have exposed cell that wax layer hides and have and directly infect characteristic; 2. the growing tip cell has outward by wax layer, is not in naked state, divides vigorously, is the good genetic recipient of susceptibility; 3. the young sprout quantity of xylophyta is big, and terminal bud growing tip blastoprolepsis develops into branch and cuttage Cheng Miao easily; The present invention innovates the live body growing tip Agrobacterium-mediated Transformation method that proposes xylophyta; Has following advantage: 1. do not rely on plant gene type and tissue culture; Overcome two big obstacles that xylophyta exists based on the genetic transformation technology of tissue culture and plant regeneration: the gene transformation obstacle that polyphenol obstruction Agrobacterium is infected and highly depend on genotypic aregeneratory is applicable to most of xylophyta kinds, strain and germ plasm resource material; 2. transformation efficiency is high, can realize high-throughout gene transformation; 3. simple to operate, technical key point is grasped easily, and success ratio is big, with low cost relatively, is fit to large-scale application; 4. with live body growing tip cell as genetic recipient, the vegetative propagation mode is adopted in the breeding of positive branch, has avoided environmental exposures such as gene is elegant as much as possible; This method is applicable to most of xylophytas such as tea tree, especially the full xylophyta of terminal bud.
The mechanism of inferring the live body growing tip Agrobacterium-mediated Transformation method of xylophyta of the present invention is: 1. outermost two confluent monolayer cells of growing tip are made a living and are grown the source of awl differentiation, have extremely strong growth and differentiation capability; Though 2. there is the leaf primordium parcel growing tip outside, does not have stratum corneum closely to cover, Agrobacterium bacterium fluid power penetrates into the growing tip place enough easily, and the exposed cell that the growing tip cell hides for no wax layer, Agrobacterium can directly be contaminated; 3. growing tip is the core position of plant, has best nutritional condition and physiological situation, as long as when carrying out live body gene transformation operation, excessively do not injure growing tip, then transforms the growth and the well differentiated of back growing tip cell.
Description of drawings
Fig. 1 is that the kantlex of resistance branch is identified; Wherein the square frame place is the kantlex treatment zone of resistance branch half ripe blade; Circled is the kantlex treatment zone of non-resistance branch half ripe blade; It is thus clear that the kantlex treatment zone of resistance branch half ripe blade has only pin hole, no chlorisis, no macula lutea, and the kantlex treatment zone chlorisis of non-resistance branch half ripe blade and flavescence;
Fig. 2 is that the GUS dyeing of resistance branch is identified, wherein 31 negative contrasts, and 32,33 is the resistance branch, and ck is non-resistance branch, and the GUS dyeing of visible resistance branch is blue;
Fig. 3 is that the PCR of resistance branch identifies that wherein the M swimming lane is a DL2000 dna molecular amount standard, and-swimming lane is the non-transgenic plant, the positive contrast of+swimming lane (Agrobacterium engineering bacteria), and the 1-6 swimming lane is that kantlex is identified and the transfer-gen plant that is positive is identified in GUS dyeing.
Embodiment
In order to make the object of the invention, technical scheme and advantage clearer, will combine accompanying drawing below, the preferred embodiments of the present invention are carried out detailed description.In view of tea tree belongs to the perennial woody plant, and polyphenol content is very high, is the typical case's representative in the xylophyta.Therefore; The preferred embodiments of the present invention are research object with the tea tree; Adopt live body growing tip Agrobacterium-mediated Transformation method to transform 5 genotype of tea tree reporter gene gus and amount to about 100,000 growing tip; Finally identify and the PCR evaluation, obtain transfer-gen plant 32 strains altogether through kalamycin resistance evaluation, GUS dyeing.The live body growing tip Agrobacterium-mediated Transformation method of other xylophyta can be with reference to the live body growing tip Agrobacterium-mediated Transformation method of tea tree, and basic step is consistent, only needs relevant parameter and material suitably optimized and revised to get final product.
One, live body growing tip Agrobacterium-mediated Transformation method and the condition optimizing thereof of tea tree
1, the structure of Agrobacterium engineering bacteria
Adopt the heat shock conversion method that plant binary expression vector pCAMBIA2301 is changed among the agrobacterium tumefaciens bacterial strain LBA4404, obtain the Agrobacterium engineering bacteria.
Marker gene transforms and non-transformed cell in order to distinguish in the genetic transformation process of plant, must import transformant jointly with goal gene.The marker gene of widespread use at present has herbicide resistance gene and antibiotics resistance gene.The coded product of resistant maker gene normally decomposes weedicide or antibiotic enzyme; Can give transformant weedicide or antibiotics resistance, thereby transformant can be grown and non-transformed cell is killed or grow and be suppressed in the presence of finite concentration weedicide or microbiotic.In the present invention, selective agent can be selected according to the marker gene in the goal gene recombinant expression vector, and the optimal concentration of selective agent is promptly screened to press and looked different plants and have than big-difference, can confirm through experiment.
Contain the beta-glucosiduronatase gene that intron (gus) of CaMV35S startup and the neomycin phosphotransferase gene (npt II) that CaMV35S starts in the pCAMBIA2301 carrier; The coded product of npt II gene has resistance to kantlex; Therefore, present embodiment is selective agent with the kantlex.
2, the cultivation of Agrobacterium engineering bacteria
Picking Agrobacterium engineering bacteria list colony inoculation is in the YEP liquid nutrient medium that contains the Rifampin that kantlex that concentration is 100mg/L, Streptomycin sulphate that concentration is 25mg/L and concentration is 40mg/L; In temperature is that 28 ℃, hunting speed are shaking culture under the condition of 250rpm, as bacterium liquid OD
600Value reaches at 0.8 o'clock, is centrifugal 10 minutes of 5000rpm with the rotating speed, collects thalline, uses that to contain concentration be that the MS liquid nutrient medium of Syringylethanone of 100 μ mol/L is resuspended, is used to prepare Agrobacterium engineering bacteria infiltration conversion fluid.
What 3, the growing tip screening was pressed confirms
On the terminal bud growing tip of outdoor planting tea shoot injection concentration be respectively 50,100,200,300,400,500, the kantlex aqueous solution of 600mg/L; Observed the reaction of growing tip in back 15 days and 30 days respectively at handling, press to confirm screening to the kantlex of different concns.
4, the preparation of Agrobacterium engineering bacteria infiltration conversion fluid
In the kantlex that uses the resuspended Agrobacterium engineering bacteria of MS liquid nutrient medium that contains Syringylethanone to be inoculated in to contain concentration as 100mg/L, the Streptomycin sulphate and concentration LB or MS liquid nutrient medium as the Rifampin of 40mg/L of concentration as 20mg/L; In temperature is that 28 ℃, hunting speed are shaking culture under the condition of 250rpm, as bacterium liquid OD
600Value reaches at 0.6～0.8 o'clock, adds kantlex, sucrose, SilwetL-77 and polysorbas20 by following different ingredients respectively, and preparation Agrobacterium engineering bacteria infiltration conversion fluid is investigated the influence of Agrobacterium engineering bacteria infiltration conversion fluid to live body growing tip transformation efficiency.
The prescription of Agrobacterium engineering strain infiltration conversion fluid is (concentration of each reagent is final concentration) as follows:
A: Agrobacterium engineering bacteria bacterium liquid+LB liquid nutrient medium;
B: Agrobacterium engineering bacteria bacterium liquid+LB liquid nutrient medium+concentration is the kantlex of 300mg/L;
C: Agrobacterium engineering bacteria bacterium liquid+LB liquid nutrient medium+concentration is that sucrose+concentration expressed in percentage by volume of 100g/L is that 0.05% Silwet L-77+ concentration expressed in percentage by volume is 0.03% polysorbas20;
D: Agrobacterium engineering bacteria bacterium liquid+LB liquid nutrient medium+concentration is that kantlex+concentration of 300mg/L is that sucrose+concentration expressed in percentage by volume of 100g/L is that 0.05% Silwet L-77+ concentration expressed in percentage by volume is 0.03% polysorbas20;
E: Agrobacterium engineering bacteria bacterium liquid+MS liquid nutrient medium+concentration is that kantlex+concentration of 300mg/L is that sucrose+concentration expressed in percentage by volume of 100g/L is that 0.05% Silwet L-77+ concentration expressed in percentage by volume is 0.03% polysorbas20.
5, the conversion of live body growing tip inoculation
Under different weather condition, transform inoculation respectively: 1st, 2 groups of cloudy daies with no rain are main; The 3rd group is main with fine day; With injector for medical purpose Agrobacterium engineering bacteria infiltration conversion fluid 10～50 μ L are injected to rapidly on the growing tip in each young sprout terminal bud bud bud of tea tree; Do not damage during injection or minor injury's growing tip only, and do distinguishing mark with permanent pen stem between transformant second blade face and second and third leaf; Transform and inoculate preceding 3 days with the strong calico covering young sprout terminal bud of suction moisture retention; And regularly squirt cotton and preserve moisture with clear water; Respectively at 10 of every mornings and point measurement leaf layer temperature and humidity and calculate and transform preceding 3 days MV in afternoons 3, investigate the influence of the average temperature of leaf layer, humidity to live body growing tip transformation efficiency.
6, the common cultivation of growing tip and Agrobacterium engineering bacteria
Covering sunshade in difference respectively preserves moisture and carry out common cultivation under handling: the 1st group does not cover the sunshade processing of preserving moisture; 2nd, 3 groups after transforming inoculation and accomplishing; Cover the conversion terminal bud rapidly with the strong calico of suction moisture retention earlier; The 2nd group covers 90% black sunshade net covering 50% green sunshade net, the 3rd group on the calico more again on cotton, cover the sunshade common cultivation 6 days of preserving moisture, and using the mass and size percentage concentration in wherein preceding 2 days is that 1 ‰ aqueous sucrose solution squirts cotton and preserves moisture; The back squirted cotton with clear water in 4 days and preserves moisture, and the different covering of investigation sunshades are preserved moisture and handled the influence to live body growing tip transformation efficiency.
7, the screening of resistant buds
Under difference screening press strip spare, carry out the screening of resistant buds respectively: the 1st group is not replenished screening and presses; The 2nd group is replenished screening and presses; After cultivating end altogether, injection concentration is the kantlex aqueous solution 50 μ L of 300mg/L on each conversion terminal bud growing tip, suppresses the division of non-transformed cell in the growing tip; Make the transformant proper splitting and be divided into resistant buds, investigate different screenings and press influence live body growing tip transformation efficiency.
8, the cultivation of resistance branch
Carry out management such as field fertilization and insect pest preventing and controlling by ordinary method, in time cut and newly grow but do not have the non-processing branch of transformation marker, prune per 1～February 1 time, make the resistant buds preferred growth, form resistance young sprout and resistance branch.
9, the kalamycin resistance of resistance branch is identified
What (1), the screening of half ripe blade was pressed confirms
On the half ripe blade of outdoor planting tea shoot, directly drip or the punching back drips that concentration is respectively 4,8,10,12,14, the kantlex aqueous solution of 16g/L; And preserve moisture with cotton balls and to shelter from heat or light; Observed of the reaction of half ripe blade in back 15 days and 30 days respectively at handling, press to confirm screening to the kantlex of different concns.
(2), the kalamycin resistance of resistance branch is identified
Treat that the resistance branch grows to sufficient length; It is exemplary functions leaf mid-term stage that blade on it is the half ripe state, selects the cloudy day of no rain, and dropping concentration in punching back is the kantlex aqueous solution 200 μ L of 15g/L on blade; Added in second day 1 time; Handle the colour-change situation of observing treatment zone on the blade after a few days, the extremely slight person of no chlorisis, no macula lutea or degree promptly has kalamycin resistance, and chlorisis flavescence person does not then have kalamycin resistance (as shown in Figure 1).
10, the GUS of resistance branch dyeing is identified
When treating that the resistance branch grows to sufficient length, clip 0.5cm
2Tender leaf; Immerse dyeing in the GUS dyeing working fluid (it is active to suppress in the plant materials GUS to contain concentration expressed in percentage by volume and be 20% methyl alcohol); 37 ℃ of incubated overnight; To use concentration expressed in percentage by volume again be 90% aqueous ethanolic solution decolouring rinsing 2 times eliminating chlorophyllous interference, and whether tissues observed dyeing situation successfully changes in the tea tree genome and express to confirm the gus gene.Positive staining is blue (as shown in Figure 2).
11, the PCR of resistance branch identifies
Get the functional leaf of resistance branch, extract genomic dna with conventional CTAB method; With this genomic dna is that template, 5 '-ctgaactggcagactatcccg-3 ' and 5 '-gtccgcatcttcatgacgacc-3 ' is that the upstream and downstream primer carries out the PCR evaluation; The PCR system is that TV is the standard Taq PCR system of 25 μ L, wherein contains genomic dna 2 μ l; PCR cycling condition parameter is: 94 ℃ of preparatory sex change 4 minutes, and 94 ℃ of sex change are 1 minute then, 60 ℃ of annealing 1 minute, 72 ℃ were extended 1 minute, totally 35 circulations, last 72 ℃ were extended 10 minutes, 25 ℃ of insulations 10 minutes.Get the PCR product and carry out agarose gel electrophoresis, have unique specific band person of 489bp to be judged to the positive, do not have this band person and be judged to feminine gender (as shown in Figure 3).
(2), the genotypic live body growing tip of different tea trees Agrobacterium-mediated Transformation
Adopt above-mentioned live body growing tip Agrobacterium-mediated Transformation method and optimal conditions thereof; The present invention has carried out Fuding white tea, No. 1, the tea in Chongqing, two strains of Chongqing No. 23 kinds of tea totally 5 genotypic live body growing tip Agrobacterium-mediated Transformation altogether; The tea that wherein changes is large leaf type No. 1; Fuding white tea and strain are 1 for middle blade profile, and No. 2, Chongqing tea and strain are 2 to be microphyll type.
(3), statistical method
Because whole experimental size is very big, is not easy to accurate counting, historical facts or anecdotes is tested the result and is sampling evaluation value.Kalamycin resistance is identified repetition 3 times, and at least 2 times the positive person of result counts statistics, and GUS dyeing is identified and the PCR evaluation respectively repeats 2 times, and 2 times all positive person of result counts statistics.
Two, experimental result
What 1, the growing tip screening was pressed confirms
Experimental result shows that the screening of growing tip is pressed to 300mg/L, depresses in this screening, and the speed of growth of growing tip obviously is suppressed but can cause growing tip dead.
2, Agrobacterium engineering bacteria infiltration conversion fluid prescription is to the influence of live body growing tip transformation efficiency
Agrobacterium engineering bacteria infiltration conversion fluid prescription is seen table 1 to the influence of live body growing tip transformation efficiency.Can know that by table 1 Agrobacterium engineering bacteria infiltration conversion fluid has remarkably influenced to live body growing tip transformation efficiency: prescription A does not have screening pressure, permeate agent and tensio-active agent, and transformation efficiency is 0; Prescription B has screening to press but does not have permeate agent and tensio-active agent, and prescription C has permeate agent with tensio-active agent but there is not the screening pressure, and transformation efficiency is lower; The existing screening of prescription D and E is pressed has permeate agent and tensio-active agent again, and transformation efficiency is higher; Explain that screening pressure, permeate agent and tensio-active agent all are the key factors that influence live body growing tip transformation efficiency.In addition; The kinds of culture medium of infiltration conversion fluid also has considerable influence to live body growing tip transformation efficiency; The LB liquid nutrient medium is superior to the MS liquid nutrient medium, explains that the vigor that infects of in live body growing tip Agrobacterium-mediated Transformation method, guaranteeing the Agrobacterium engineering bacteria is more even more important than the comfort level that guarantees terminal bud.
Table 1 Agrobacterium engineering bacteria infiltration conversion fluid prescription is to the influence of live body growing tip transformation efficiency
Infiltration conversion fluid kalamycin resistance GUS dyeing PCR identifies
The prescription positive plant is counted positive plant and is counted the positive plant number
A 0 0 0
B 8 0 1
D 92 23 29
E 56 9 7
3, the average temperature of leaf layer, humidity are to the influence of live body growing tip transformation efficiency
The average temperature of leaf layer, humidity are seen table 2 to the influence of live body growing tip transformation efficiency.Can be known that by table 2 the 1st, 2 group of leaf layer medial temperature difference is less, all infect 23 ℃ of optimum temperutures near the Agrobacterium of bibliographical information, but medial humidity has certain difference, the 1st group the transformation efficiency that medial humidity is bigger is superior to less the 2nd group of medial humidity; The 3rd group of leaf layer medial temperature and Agrobacterium are infected optimum temperuture and differ bigger, and leaf layer medial humidity is lower, and transformation efficiency is 0; Show that leaf layer medial humidity is the important factor that influences live body growing tip transformation efficiency, higher leaf layer humidity has guaranteed that Agrobacterium engineering bacteria infiltration conversion fluid not by rapid evaporation, makes the Agrobacterium engineering bacteria have enough vigor and time that growing tip is infected.
The average temperature of table 2 leaf layer, humidity are to the influence of live body growing tip transformation efficiency
Numbering leaf layer medial temperature/humidity kalamycin resistance GUS dyeing PCR identifies
(℃/%) positive plant is counted positive plant and is counted the positive plant number
1 22.3/80 92 20 21
2 24.5/72 70 12 16
3 27.3/65 0 0 0
4, different covering sunshades are preserved moisture and are handled the influence to live body growing tip transformation efficiency.
Different covering sunshades are preserved moisture to handle table 3 are seen in the influence of live body growing tip transformation efficiency.Can know by table 3, cover sunshade and preserve moisture that to handle be the outdoor key measure that the Agrobacterium engineering bacteria transforms the environmental optima condition of keeping that preserve moisture when handling when not covering sunshade, transformation efficiency is 0; Preserve moisture when handling when adopting the green sunshade net of cotton+50% or cotton+90% black sunshade net to cover sunshade; Transformation efficiency higher and between the two difference little; Possibly be because the hydrojet moisture-keeping function of cotton, on cotton, cover 50% green sunshade net again or 90% black sunshade net is not remarkable to the influence of cotton inferior lobe layer temperature, humidity.
The different sunshading and thermal insulatings that cover of table 3 are handled the influence to live body growing tip transformation efficiency
Kalamycin resistance GUS dyeing PCR identifies
Cover the sunshade processing of preserving moisture
Positive plant is counted positive plant and is counted the positive plant number
Not covering sunshade preserves moisture and handles 000
The green sunshade net 80 15 19 of cotton+50%
Cotton+90% black sunshade net 86 17 18
5, the influence to live body growing tip transformation efficiency is pressed in different screenings
Different screenings are pressed table 4 are seen in the influence of live body growing tip transformation efficiency.Can know by table 4; Replenishing injection concentration immediately after cultivation finishes altogether is the kantlex of 300mg/L; Making growing tip between the differentiation phase, keep suitable screening presses; Help that transformant preferentially divides in the growing tip, form the resistance growing tip and also be divided into resistant buds, reduce forming chimeric possibility simultaneously.
The influence to live body growing tip transformation efficiency is pressed in the different screenings of table 4
Kalamycin resistance GUS dyeing PCR identifies
Whether replenish and select to press
Positive plant is counted positive plant and is counted the positive plant number
Replenish 148 27 31
Do not replenish 14 56
What 6, the screening of half ripe blade was pressed confirms
Experimental result shows, the half ripe blade of outdoor planting tea shoot is owing to have the thicker wax layer of one deck and receive the influence of natural lighting environment, and all kantlex concentration gradients directly drip to handle and all leaf growth produced restraining effect in 30 days; And the result of punching back dropping kantlex has differences in concentration gradient; Handling the kantlex of back 15 days concentration below 10g/L handles leaf growth unrestraint effect; Handle back 30 days concentration kantlex below 8g/L and handle leaf growth unrestraint effect, concentration is that the kantlex of 12g/L handles that the leaf but the later stage can not bore a hole macula lutea appears, in respective regions on 15～30 days blades in back; And yellow appears in respective regions on 15～30 days blades in concentration to be the kantlex of 16g/L handle back; Later stage perforation occurs but does not fall leaf, therefore, confirms that the screening pressure of half ripe blade is 12～15g/L.
7, different tea tree genotype are to the influence of live body growing tip transformation efficiency
Different tea tree genotype are seen table 5 to the influence of live body growing tip transformation efficiency.Can know that by table 5 different tea tree genotype have certain influence to live body growing tip transformation efficiency, the transformation efficiency of large leaf type and middle blade profile higher and between the two difference little, the transformation efficiency of microphyll type is relatively low.Analyzing its reason possibly be: the 1. influence of growing tip internal substance: bibliographical information, the bacterinertness of tea tree tea-polyphenol are the important factors that reduces transformation efficiency.The polyphenol content of three kinds of blade profile tea trees of this experiment is followed successively by: large leaf type＞middle blade profile＞microphyll type; But the transformation efficiency of the large leaf type that polyphenol content is high is higher than the low microphyll type of polyphenol content; Possibly be because the difference of three kinds of blade profile tea tree polyphenol contents does not have remarkably influenced in spring to the transformation efficiency of live body growing tip, and large leaf type is owing to have bigger terminal bud and be easy to transform successfully.2. the influence of bud type: three kinds of blade profile tea tree middle and later periods in spring of this experiment size and full degree of young sprout terminal bud is followed successively by: large leaf type＞middle blade profile＞microphyll type; Just become positive correlation, explain that the size of young sprout terminal bud and full degree possibly be the important factors that influences live body growing tip transformation efficiency with live body growing tip transformation efficiency.Big and full bud head can hold more Agrobacterium engineering bacteria infiltration conversion fluid, thereby can guarantee enough engineering bacteria amounts, and can make engineering bacteria keep the vigor of longer time.In addition, the injector for medical purpose of same model is less relatively to the injury of large leaf type growing tip, thereby makes its transformation efficiency higher.
The different tea tree genotype of table 5 are to the influence of live body growing tip transformation efficiency
Tea tree genotype kalamycin resistance positive plant is counted GUS stained positive plant and is counted PCR evaluation positive plant number
Large leaf type 82 15 18
Middle blade profile 70 15 16
Microphyll type 10 23
Above embodiment is only unrestricted in order to technical scheme of the present invention to be described; Although through invention has been described according to the preferred embodiments of the present invention; But those of ordinary skill in the art is to be understood that; Can make various changes to it in form with on the details, and the spirit and scope of the present invention that do not depart from appended claims and limited.
Sequence table
< 110>Chongqing Agricultural Academy of Science
< 120>the live body growing tip Agrobacterium-mediated Transformation method of xylophyta
<160>2
<210>1
<211>21
<212>DNA
< 213>artificial sequence
<220>
< 223>upstream primer of the PCR of resistance branch evaluation
<400>1
ctgaactggc agactatccc g 21
<210>2
<211>21
<212>DNA
< 213>artificial sequence
<220>
< 223>downstream primer of the PCR of resistance branch evaluation
<400>2
gtccgcatct tcatgacgac c 21
Claims (5)
1. the live body growing tip Agrobacterium-mediated Transformation method of xylophyta, it is characterized in that: said xylophyta is a tea tree, said live body growing tip Agrobacterium-mediated Transformation method may further comprise the steps:
The preparation of a, Agrobacterium engineering bacteria infiltration conversion fluid: the Agrobacterium engineering bacteria that will contain goal gene is inoculated in the liquid nutrient medium that contains the screening pressure and cultivates, as bacterium liquid OD
600Value reaches at 0.5～1.0 o'clock, in bacterium liquid, adds screening and presses, and add permeate agent and tensio-active agent, is mixed with Agrobacterium engineering bacteria infiltration conversion fluid;
The conversion of b, live body growing tip inoculation: in leaf layer medial temperature is that 18～26 ℃, medial humidity are under 70～80% the condition, and Agrobacterium engineering bacteria infiltration conversion fluid is injected on the growing tip in the xylophyta young sprout terminal bud bud bud;
The common cultivation of c, growing tip and Agrobacterium engineering bacteria: the young sprout terminal bud after will transforming cover the suction material which can retain moisture and the condition of the cooling moisture-retaining that shelters from heat or light with the sunshade net under cultivated altogether 2～10 days, control leaf layer medial temperature is that 18～26 ℃, medial humidity are 70～80%;
The screening of d, resistant buds: after cultivating end altogether, on the growing tip of young sprout terminal bud, add screening and press, suppress the division of non-transformed cell in the growing tip, make the transformant proper splitting and be divided into resistant buds;
The cultivation of e, resistance branch: carry out field fertilization and insect pest preventing and controlling management by ordinary method, in time cut the branch that unconverted is handled, make resistant buds grow into the resistance branch;
The evaluation of f, transfer-gen plant: the antagonism branch carries out transgenic and identifies that the plant that the result is positive is transfer-gen plant.
2. according to the live body growing tip Agrobacterium-mediated Transformation method of the said xylophyta of claim 1, it is characterized in that: the terminal bud of said xylophyta is full.
3. according to the live body growing tip Agrobacterium-mediated Transformation methods of claim 1 or 2 said xylophytas; It is characterized in that: said step a is inoculated in the Agrobacterium engineering bacteria that contains goal gene to contain in the LB liquid nutrient medium that screens pressure, Streptomycin sulphate and Rifampin to cultivate, as bacterium liquid OD
600Value reaches at 0.6～0.8 o'clock; In bacterium liquid, adding screening presses; And to add concentration be that sucrose, the concentration expressed in percentage by volume of 100g/L is that 0.02～0.05% SilwetL-77 and concentration expressed in percentage by volume are 0.01～0.05% tween, is mixed with Agrobacterium engineering bacteria infiltration conversion fluid.
4. according to the live body growing tip Agrobacterium-mediated Transformation method of claim 1 or 2 said xylophytas, it is characterized in that: said step b transforms inoculation full period at xylophyta young sprout terminal bud, and does not damage when transforming inoculation or minor injury's growing tip only.
5. according to the live body growing tip Agrobacterium-mediated Transformation methods of claim 1 or 2 said xylophytas; It is characterized in that: said step c covers the young sprout terminal bud after transforming with the strong cotton of suction moisture retention earlier; On cotton, cover 50%～90% sunshade net again; Growing tip and Agrobacterium engineering bacteria were cultivated 6 days altogether, squirted cotton with aqueous sucrose solution in wherein preceding 2 days to preserve moisture, back 4 days waters squirt cotton and preserve moisture.
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| CN108531507A (en) * | 2018-04-16 | 2018-09-14 | 广东省农业科学院果树研究所 | A kind of simple and quick citrus marker-free transgenic method |
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| CN101225384A (en) * | 2008-02-04 | 2008-07-23 | 山西省农业生物技术研究中心 | Agrobacterium-mediated woody plants leaf mud in-situ gene transformation method |
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| CN101225384A (en) * | 2008-02-04 | 2008-07-23 | 山西省农业生物技术研究中心 | Agrobacterium-mediated woody plants leaf mud in-situ gene transformation method |
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