CN104140978A - Genetic transformation method using horseradish seed leaves as explant - Google Patents
Genetic transformation method using horseradish seed leaves as explant Download PDFInfo
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- CN104140978A CN104140978A CN201410353144.5A CN201410353144A CN104140978A CN 104140978 A CN104140978 A CN 104140978A CN 201410353144 A CN201410353144 A CN 201410353144A CN 104140978 A CN104140978 A CN 104140978A
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Abstract
The invention discloses a genetic transformation method using horseradish seed leaves as an explant. The method comprises the steps of (1) activated cultivation of agrobacterium strains, (2) obtaining of horseradish aseptic seedlings, (3) precultivation of the horseradish seed leaves, (4) genetic transformation of the horseradish seed leaves, and (5) rapid proliferation of horseradish hairy roots. According to the genetic transformation method, genetic transformation is achieved when agrobacterium rhizogenes successfully induce the horseradish seed leaves, and a large number of hairy roots which contain secondary metabolites, can grow rapidly and are stable genetically are obtained.
Description
Technical field
The present invention relates to horseradish genetic transfoumation field, disclose especially a kind of genetic transforming method that horseradish cotyledon is explant of take.
Background technology
Horseradish (Wasabi japonica Matsum) has another name called Euterma Wasabi, mountain dish, is the perennial half cloudy raw herbaceous plant of Cruciferae Eutrema.The material that contains a kind of mustard seed glycoside in horseradish plant, when plant materials is grated, this material is hydrolyzed and forms isosulfocyanate compound under the effect of myrosinase, wherein especially the highest with propenyl isothiocyanate content, accounts for 89%～94%; In addition, horseradish plant also contains the trace element of a large amount of amino acid and various human body needs.The amino acid and the trace element that contain abundant isosulfocyanate compound and a large amount of human body needs due to horseradish, it not only has strong sharp flavor and the special pungent taste of perfume (or spice), but also there is the multiple effects such as immunomodulatory, antibacterial, anticancer, anti-oxidant, platelet aggregation-against, be the very rare and precious medicinal and edible plant of generally acknowledging in the world, be called as " green gold ".
Apply and form a sharp contrast widely with it, horseradish resource is very limited, this is because growing of horseradish plant has very harsh requirement to environment, and it is that 1000～2000 meters, temperature range are brook growth in the nice and cool woods of the humidity of 8～l8 ℃ that horseradish is suitable in sea level elevation.Once surpassing 30 ℃, horseradish fertility temperature easily there is soft rot, black heart, Powdery Mildew and virus disease, wherein serious with soft rot and black disease; But during lower than-3 ℃, be subject to again freeze injury; In addition, horseradish setting percentage is low, and the seed dormancy phase is long, and its milpa can not be planted continuously; A common strain horseradish plant was through the plantation of 18 months, and the rhizome of results is on average less than 200g.Therefore, only depending at present the limited plantation amount of horseradish, is far can not meet the demand in market.
Patent CN1204453A discloses a kind of rapid breeding method of horseradish seedling, comprise: under aseptic condition, cut the lateral bud on horseradish terminal bud or root stock, by the change to the selection of substratum, hormone combination, culture condition, the fast breeding cultivation, the growth of plants cultivation, root culture, the rooting culture that carry out test-tube plantlet induction, test-tube plantlet make the propagation of horseradish tissue cultured seedling rapid, its breeding coefficient can reach 4.0-5.0, the test-tube plantlet that multiplication culture forms is directly used in production, save labor, economize ground, speed fast, reduce production costs.Patent CN102668992A discloses a kind of horseradish tissue cultured seedling outside sprout-cultivating-bottle method, it utilizes tissue culture induction horseradish stem apex to obtain unrooted Shoots in vitro, after succeeding transfer culture, bud seedling is cut into simple bud through domestication, again bud seedling is inserted in soil matrix, water weekly during this time promoter and 1/2MS nutritive medium once, after insertion soil matrix 30d, can obtain good rooting efficiency.Patent CN102823491A discloses a kind of horseradish callus culture method that horseradish root is explant of take, comprise the steps: the processing of (1) explant: get the horseradish root of cutting from horseradish plant, after rinsing well with flowing water, be put in and on filter paper, blot surface-moisture, then place it in the mercuric chloride that is 0.1%～0.15% by concentration on Bechtop sterilization 5～8min, then with sterilized filter paper, blot surperficial moisture afterwards 2～5 times with aseptic water washing; (2) inducing culture of callus: the length that is 1-2cm by the horseradish undercut of having sterilized, then accesses callus culture base MS+6-BA0.5～4.0mgL
-1+ 2,4-D-1.0～6.0-mgL
-1+ IAA0.5～3.0mgL
-1in carry out inducing culture, culture condition is: 10～28 ℃ of temperature, illumination every day 4～10 hours, intensity of illumination is 600～1200lx; (3) multiplication culture of callus: the callus that step (2) is made is transferred into callus proliferated culture medium 1/2MS+6-BA0～1.0mgL
-1+ 2,4-D1.0～4.0mgL
-1+ KT0.5～1.0mgL
-1+ Sulfothiorine 0.5～4.0gL
-1in carry out multiplication culture, culture condition is: 10～28 ℃ of temperature, illumination every day 6～12 hours, intensity of illumination are 800～1500lx; The pH value of above-mentioned all substratum is 5.6～6.5, sucrose 15～30gL
-1, agar 5.0～6.0gL
-1.It,, by cultivating in access callus culture base horseradish root as explant, has successfully induced the high horseradish callus of active constituent content.Patent CN103053426A discloses a kind of horseradish seedling cultural method, comprises the following steps: the cultivation of (A) taking root: the shoot apical meristem of educating being just commissioned to train through sterilizing detoxification is transferred in the 1/2MS substratum that does not add hormone and grown; (B) transplant hardening: step (A) continued after 28～32 days, be moved in the dish of cave; (C) field planting: step (B) was transplanted hardening after 75～85 days, by finished product horseradish seedling replanting to land for growing field crops.It adopts the mode of adding root-growing agent in the substratum of multiplication culture to replace phytokinin, has overcome the problem slow, root is elongated of taking root, and has reached the object of the root length of the individual plant seedling that increases sharply in a short time.Patent CN103718963A discloses a kind of cultural method of horseradish detoxic seedling, it selects aseptic horseradish sprout is cultivated material, after alternating temperature is processed, get in its bud meristematic tissue access differentiation substratum, through the inducing culture of strong seedling culture base and root media and obtain horseradish detoxic seedling, the horseradish detoxic seedling of cultivating has fast growth, active principle content advantages of higher again.
Growing and ripe along with current biotechnology, has become study hotspot in recent years by the research that Agrobacterium rhizogenes mediated plant carries out genetic transformation at present.This be due to hairly root that Agrobacterium rhizogenes mediated plant produces have growth rapidly, inheritance stability and can independently growth on without hormone culture-medium, and it can also synthesize with former plant content quite or higher than the secondary metabolite of former plant content; Produced simultaneously hairly root also has the potentiality that are grown to serve as transgenic regenerated plant.Yet the research article that up to the present, carries out genetic transformation by Agrobacterium rhizogenes mediation horseradish plant yet there are no report.
Summary of the invention
The object of the present invention is to provide a kind of genetic transforming method that horseradish cotyledon is explant of take, by Agrobacterium rhizogenes, successfully induce horseradish cotyledon to realize genetic transformation, obtained contain in a large number secondary metabolite, growth rapidly, inheritance stability and hairly root that can Fast Growth.
The invention provides a kind of genetic transforming method that horseradish cotyledon is explant of take, comprise the steps:
Step 1, the activation culture of Agrobacterium bacterial classification: after Agrobacterium rhizogenes is rule on YEB solid medium, be placed under the temperature condition of 26～30 ℃ and carry out secretly cultivating after 24～36h, in picking list bacterium colony access YEB liquid nutrient medium, and after 1～2 day, record bacterium liquid OD in 25～30 ℃ of shaking culture
600=0.1～0.3 left and right can be used for explant and contaminates;
Step 2, the acquisition of horseradish aseptic seedling: wasabi seed is placed on Bechtop, first uses alcohol disinfecting 20～40s of 75%, then with 0.1% mercuric chloride sterilization 3～8min, after aseptic water washing 2～5 times, peel off kind of a skin and be seeded in MS+6-BA0.5～4mgL
-1+ NAA (or IAA) 0.5～2mgL
-1+ sucrose 20～50gL
-1+ agar 3.5～4.5gL
-1substratum on, and at 22～28 ℃, light intensity 1000～2000lx, cultivates under the condition of illumination every day 8～12h, obtains horseradish aseptic seedling;
Step 3, the preculture of horseradish cotyledon: cut cotyledon that horseradish aseptic seedling just launched 1-4d as explant, be inoculated on pre-culture medium, and 18～22 ℃, without carrying out the preculture of 1～3d under illumination condition;
Step 4, the genetic transformation of horseradish cotyledon: choose after preculture and to produce in horseradish cotyledon incision the explant expanding and put into the Agrobacterium bacterium liquid that step (1) activated and contaminate 1～3 minute, after explant is fully contacted with Agrobacterium, and blot after the unnecessary bacterium liquid in explant surface with aseptic filter paper, proceed to again in common substratum, under the dark culture condition of 15～22 ℃, carry out the common cultivation of 1～3d; Proceeded to subsequently containing after soaking in the microbiotic water of cefotaxime sodium, then proceeded to except carrying out degerming cultivation in bacterium culture medium, every 2～4d switching, once removed bacterium culture medium, until hairly root produces in a large number.
Step 5, the fast breeding of horseradish hairly root: the hairly root switching that cuts length and be 1.5～3cm is cultivated in hairly root fast breeding substratum.
The Agrobacterium adopting in above-mentioned steps (1) is Agrobacterium rhizogenes A4 or R1000, contaminates the bacterium liquid OD of horseradish cotyledon explant
600=0.1～0.3;
In above-mentioned steps (2), the wasabi seed after sterilization is peelled off to kind of skin and is seeded in MS+6-BA0.5～4mgL
-1+ NAA (or IAA) 0.5～2mgL
-1+ sucrose 20～50gL
-1+ agar 3.5～4.5gL
-1substratum on, and at 22～28 ℃, light intensity 1000～2000lx, cultivates under the condition of illumination every day 8～12h, obtains horseradish aseptic seedling;
In above-mentioned steps (3), be to choose cotyledon that horseradish aseptic seedling just launched 1-4d as explant;
In above-mentioned steps (3), pre-culture medium is MS+2,4-D1～2mgL
-1+ KT0.1～0.5mgL
-1+ VITAMIN B4 10～20mgL
-1+ sucrose 20～50gL
-1+ agar 5～7gL
-1;
In above-mentioned steps (3), the preculture condition of horseradish cotyledon be 18～22 ℃, without carrying out the preculture of 1～3d under illumination condition;
In above-mentioned steps (4), be to choose after preculture in horseradish cotyledon incision, to produce the explant expanding and put into the Agrobacterium bacterium liquid having activated and contaminate 1～3 minute;
Common substratum in above-mentioned steps (4) is 1/2MS+ pyrocatechol 100～200mgL
-1+ sucrose 10～15gL
-1+ agar 4～5.5gL
-1; And under the dark culture condition of 15～22 ℃, carry out the common cultivation of 1～3d;
In above-mentioned steps (4), the explant after cultivating is altogether proceeded to containing cefotaxime sodium 200～500mgL
-1microbiotic water in soak after 1～3 hour, then proceed to except bacterium culture medium be 1/2MS+IBA0.1～0.5mgL
-1+ agar 5～7gL
-1+ sucrose 15～30gL
-1+ cefotaxime sodium 200～400mgL
-1+ ceftriaxone sodium 200～400mgL
-1.
In above-mentioned steps (5), hairly root fast breeding substratum is 1/2MS+IBA0.1～0.5mgL
-1+ sucrose 10～20gL
-1+ cephamycin 100～300mgL
-1liquid nutrient medium on carry out hairly root fast breeding cultivate.
Beneficial effect of the present invention has: by Agrobacterium rhizogenes, successfully induce horseradish cotyledon to realize genetic transformation, obtained contain in a large number secondary metabolite, growth rapidly, inheritance stability and hairly root that can Fast Growth, for the quick production of horseradish containing plant effective component with obtain transgenic regenerated plant the important channel of is provided.
Embodiment
By specific embodiments of the invention given below and comparative example, can further be well understood to the present invention, but they not limitation of the invention.The part not describing in detail in specific embodiment and comparative example is to adopt prior art, known technology means and industry standard to obtain.
Except as otherwise noted, the percentage ratio adopting in the present invention is weight percentage.
embodiment 1
The genetic transformation that to implement as follows to take horseradish cotyledon be explant:
(1) activation culture of bacterial classification:
After Agrobacterium rhizogenes A4 is rule on YEB solid medium, be placed under the temperature condition of 28 ℃ and secretly cultivate after 30h, in picking list bacterium colony access YEB liquid nutrient medium, and after 2 days, record bacterium liquid OD in 28 ℃ of shaking culture
600=0.2 left and right can be used for explant and contaminates.
(2) acquisition of aseptic seedling:
Wasabi seed is placed on Bechtop, first uses 75% alcohol disinfecting 30s, then with 0.1% mercuric chloride sterilization 5min, after aseptic water washing 4 times, peel off kind of a skin and be seeded in MS+6-BA2mgL
-1+ NAA or IAA1mgL
-1+ sucrose 40gL
-1+ agar 4gL
-1substratum on, and at 24 ℃, light intensity 1500lx, cultivates under the condition of illumination every day 10h, cultivates and within 1 week, obtains horseradish aseptic seedling;
(3) preculture of horseradish cotyledon:
Cut 2 cotyledons of the firm 3d of expansion in horseradish aseptic seedling as explant, be inoculated in MS+2,4-D1.5mgL
-1+ KT0.3mgL
-1+ VITAMIN B4 15mgL
-1+ sucrose 30gL
-1+ agar 6gL
-1pre-culture medium on, and 20 ℃, without carrying out the preculture of 2d under illumination condition;
(4) genetic transformation of horseradish cotyledon:
After choosing preculture, in horseradish cotyledon incision, produce the explant expanding, put into bacterium liquid OD
600in=0.2 Agrobacterium bacterium liquid, contaminate 2 minutes, note therebetween shaking, after explant is fully contacted with Agrobacterium, and blot after the unnecessary bacterium liquid in explant surface with aseptic filter paper, then proceed to 1/2MS+ pyrocatechol 150mgL
-1+ sucrose 12gL
-1+ agar 4.5gL
-1common substratum in, and under the dark culture condition of 18 ℃, carry out the common cultivation of 2d; Proceeded to subsequently containing cefotaxime sodium 300mgL
-1microbiotic water in soak after 2 hours, then proceed to 1/2MS+IBA0.3mgL
-1+ agar 5.5gL
-1+ sucrose 20gL
-1+ cefotaxime sodium 300mgL
-1+ ceftriaxone sodium 300mgL
-1in substratum, continuing degerming cultivates; Every 3d switching, once except bacterium culture medium, after cultivating 30 days, adding up hairly root transformation efficiency is 28.91%.
(5) fast breeding of horseradish hairly root:
The hairly root that cuts length and be 2cm is transferred into 1/2MS+IBA0.3mgL
-1+ sucrose 15gL
-1+ cephamycin 200mgL
-1liquid nutrient medium on carry out hairly root fast breeding cultivate; The propagation multiple of adding up afterwards hairly root in cultivation for 25 days is 4.58.
embodiment 2
The process of this embodiment is substantially the same manner as Example 1, and its difference is:
In step (1), the Agrobacterium rhizogenes of contaminating horseradish cotyledon explant is used instead as R1000.Is 26.73% at step (4) horseradish cotyledon except adding up hairly root transformation efficiency after cultivating 30 days in bacterium culture medium.
embodiment 3
The process of this embodiment is substantially the same manner as Example 1, and its difference is:
In step (1), the Agrobacterium rhizogenes A4 bacterial concentration OD of cultivation
600=0.1 Agrobacterium bacterium immersion is dyed horseradish cotyledon explant.Is 21.71% at step (4) horseradish cotyledon except adding up hairly root transformation efficiency after cultivating 30 days in bacterium culture medium.
In step (1), the Agrobacterium rhizogenes A4 bacterial concentration OD of cultivation
600=0.3 Agrobacterium bacterium immersion is dyed horseradish cotyledon explant.Is 27.34% at step (4) horseradish cotyledon except adding up hairly root transformation efficiency after cultivating 30 days in bacterium culture medium.
embodiment 4
The process of this embodiment is substantially the same manner as Example 1, and its difference is:
In step (3), the preculture time of horseradish cotyledon changes 1d into.Is 20.71% at step (4) horseradish cotyledon except adding up hairly root transformation efficiency after cultivating 30 days in bacterium culture medium.
In step (3), the preculture time of horseradish cotyledon changes 3d into.Is 23.43% at step (4) horseradish cotyledon except adding up hairly root transformation efficiency after cultivating 30 days in bacterium culture medium.
embodiment 5
The process of this embodiment is substantially the same manner as Example 1, and its difference is:
In step (3), choose the cotyledon of the firm 1d of expansion in horseradish aseptic seedling as explant, change and be inoculated in MS+2,4-D1mgL
-1+ KT0.5mgL
-1+ VITAMIN B4 10mgL
-1+ sucrose 50gL
-1+ agar 7gL
-1pre-culture medium in, is 21.45% at step (4) horseradish cotyledon except adding up hairly root transformation efficiency after cultivating 30 days in bacterium culture medium.
In step (3), choose the cotyledon of the firm 4d of expansion in horseradish aseptic seedling as explant, change and be inoculated in MS+2,4-D2mgL
-1+ KT0.1mgL
-1+ VITAMIN B4 20mgL
-1+ sucrose 20gL
-1+ agar 5gL
-1pre-culture medium in, is 24.15% at step (4) horseradish cotyledon except adding up hairly root transformation efficiency after cultivating 30 days in bacterium culture medium
embodiment 6
The process of this embodiment is substantially the same manner as Example 1, and its difference is:
In step (4), after choosing preculture, in horseradish cotyledon incision, produce the explant expanding, put into agrobacterium liquid immerged time and change 3 minutes into, incubation time changes 3 days into altogether, at step (4) horseradish cotyledon, except in bacterium culture medium, cultivating after 30 days, statistics hairly root transformation efficiency is 21.31%.
In step (4), after choosing preculture, in horseradish cotyledon incision, produce the explant expanding, put into agrobacterium liquid immerged time and change 1 minute into, incubation time changes 1 day into altogether, at step (4) horseradish cotyledon, except in bacterium culture medium, cultivating after 30 days, statistics hairly root transformation efficiency is 25.13%.
embodiment 7
The process of this embodiment is substantially the same manner as Example 1, and its difference is:
Change the common substratum in step (4) into 1/2MS+ pyrocatechol 100mgL
-1+ sucrose 10gL
-1+ agar 4gL
-1; And under the dark culture condition of 15 ℃, carry out the common cultivation of 1d; After horseradish Cotyledon culture 30 days, statistics hairly root transformation efficiency is 22.61%.
Change the common substratum in step (4) into 1/2MS+ Catechol 2 00mgL
-1+ sucrose 15gL
-1+ agar 5.5gL
-1; And under the dark culture condition of 15 ℃, carry out the common cultivation of 1d; After horseradish Cotyledon culture 30 days, statistics hairly root transformation efficiency is 19.87%.
embodiment 8
The process of this embodiment is substantially the same manner as Example 1, and its difference is:
The bacterium culture medium that removes in step (4) changes 1/2MS+IBA0.5mgL into
-1+ agar 5gL
-1+ sucrose 15gL
-1+ cefotaxime sodium 400mgL
-1+ ceftriaxone sodium 200mgL
-1; After horseradish Cotyledon culture 30 days, statistics hairly root transformation efficiency is 25.54%.
The bacterium culture medium that removes in step (4) changes 1/2MS+IBA0.1mgL into
-1+ agar 7gL
-1+ sucrose 30gL
-1+ cefotaxime sodium 200mgL
-1+ ceftriaxone sodium 400mgL
-1; After horseradish Cotyledon culture 30 days, statistics hairly root transformation efficiency is 20.84%.
embodiment 9
The process of this embodiment is substantially the same manner as Example 1, and its difference is:
In step (5), horseradish hairly root proliferated culture medium changes 1/2MS+IBA0.1mgL into
-1+ sucrose 10gL
-1+ cephamycin 100mgL
-1.The propagation multiple of adding up afterwards hairly root in cultivation for 25 days is 3.42.
embodiment 10
The process of this embodiment is substantially the same manner as Example 1, and its difference is:
In step (5), horseradish hairly root proliferated culture medium changes 1/2MS+IBA0.5mgL into
-1+ sucrose 10gL
-1+ cephamycin 300mgL
-1.The propagation multiple of adding up afterwards hairly root in cultivation for 25 days is 3.89.
In step (5), horseradish hairly root proliferated culture medium changes 1/2MS+IBA0.1mgL into
-1+ sucrose 20gL
-1+ cephamycin 100mgL
-1.The propagation multiple of adding up afterwards hairly root in cultivation for 25 days is 3.27.
comparative example 1
The process of this comparative example is substantially the same manner as Example 1, and its difference is:
In step (1), the Agrobacterium rhizogenes of contaminating horseradish cotyledon explant is used instead as Ri15734.Is 0% at step (4) horseradish cotyledon except adding up hairly root transformation efficiency after cultivating 30 days in bacterium culture medium.
comparative example 2
The process of this comparative example is substantially the same manner as Example 1, and its difference is:
In step (1), use A4 bacterial concentration instead OD
600=0.6 contaminates horseradish cotyledon explant.After step (4) is cultivated 30 days, adding up hairly root transformation efficiency is 5.63%.This is mainly because horseradish cotyledon explant is more responsive to the A4 bacterium liquid of high density, by concentration, is being OD
600=0.6 bacterium immersion is dyed after explant, causes horseradish cotyledon to occur the reason of serious brownization.
comparative example 3
The process of this comparative example is substantially the same manner as Example 1, and its difference is:
The preculture step of cancellation step (3), direct employing does not produce through pre-incubated horseradish cotyledon incision the explant expanding and carries out During Agrobacterium experiment.At step (4) horseradish Cotyledon culture, after 30 days, statistics hairly root transformation efficiency is 4.89%.This just illustrates in the process of the genetic transformation of Agrobacterium, the explant transforming, after the preculture of certain hour is processed, can be adjusted to the explant somatocyte that will transform to the state of optimum Agrobacterium Induction Transformation well.
comparative example 4
The process of this comparative example is substantially the same manner as Example 1, and its difference is:
In step (3), reelect for horseradish aseptic seedling, launched 8d cotyledon as explant, after step (4) is cultivated 30 days, statistics hairly root transformation efficiency is 8.43%.
comparative example 5
The process of this comparative example is substantially the same manner as Example 1, and its difference is:
In step (3), remove the VITAMIN B4 of adding in pre-culture medium, after step (4) is cultivated 30 days, adding up hairly root transformation efficiency is 10.41%, illustrates that VITAMIN B4 has the effect of adjusting significantly horseradish cotyledon explant cell generation active state, more easily transforms successfully Agrobacterium.
comparative example 6
The process of this comparative example is substantially the same manner as Example 1, and its difference is:
Preculture condition in step (3) is changed at light intensity 1500lx, under the condition of illumination every day 10h, cultivate, in step (4), after cultivating 30 days, statistics hairly root transformation efficiency is 10.24%.
comparative example 7
The process of this comparative example is substantially the same manner as Example 1, and its difference is:
In step (4), horseradish cotyledon explant is put into Agrobacterium bacterium liquid immerged time and change 10 minutes into, incubation time changes 5 days into altogether, and at step (4) horseradish Cotyledon culture, after 30 days, statistics hairly root transformation efficiency is 3.69%.Make discovery from observation, horseradish cotyledon immerged time in Agrobacterium bacterium liquid is long, and cotyledon is subject to compared with grievous injury, also causes the cotyledon degerming after conversion more difficult simultaneously.
comparative example 8
The process of this comparative example is substantially the same manner as Example 1, and its difference is:
In step (4), the pyrocatechol adding in common substratum is removed, and change at light intensity 2000lx, under the condition of illumination every day 8h, cultivate and carry out common cultivation; After horseradish Cotyledon culture 30 days, statistics hairly root transformation efficiency is 9.59%.
comparative example 9
The process of this comparative example is substantially the same manner as Example 1, and its difference is:
Common culturing step in cancellation step (4).Cultivating after 30 days except in bacterium culture medium of step (4) horseradish cotyledon, statistics hairly root transformation efficiency is 2.81%.Explanation thus, culturing step plays a part to do important in the genetic transformation process of horseradish cotyledon altogether.
comparative example 10
The process of this comparative example is substantially the same manner as Example 1, and its difference is:
In above-mentioned steps (4), the explant after cultivating altogether directly proceeded to except bacterium culture medium, and cancel the treating processes in microbiotic water; After horseradish Cotyledon culture 30 days, statistics hairly root transformation efficiency is 14.23%.
comparative example 11
The process of this comparative example is substantially the same manner as Example 1, and its difference is:
The bacterium culture medium that removes in step (4) changes MS+IBA0.5mgL into
-1+ agar 6gL
-1+ sucrose 20gL
-1+ cefotaxime sodium 200mgL
-1; After horseradish Cotyledon culture 30 days, statistics hairly root transformation efficiency is 12.48%.
comparative example 12
The process of this comparative example is substantially the same manner as Example 1, and its difference is:
The bacterium culture medium that removes in step (4) changes MS+IBA0.5mgL into
-1+ agar 6gL
-1+ sucrose 20gL
-1+ ceftriaxone sodium 200mgL
-1; After horseradish Cotyledon culture 30 days, statistics hairly root transformation efficiency is 7.89%.
comparative example 13
The process of this comparative example is substantially the same manner as Example 1, and its difference is:
In step (5), the hairly root that cuts length and be 0.5cm carries out multiplication culture; The propagation multiple of adding up afterwards hairly root in cultivation for 25 days is 2.18.
comparative example 14
The process of this comparative example is substantially the same manner as Example 1, and its difference is:
In step (5), the cephamycin adding in hairly root fast breeding substratum is removed, the propagation multiple of adding up afterwards hairly root in cultivation for 25 days is 2.01.
comparative example 15
The process of this comparative example is substantially the same manner as Example 1, and its difference is:
In step (5), change hairly root fast breeding substratum into 1/2MS+IBA0.1mgL
-1+ sucrose 10gL
-1+ cephamycin 500mgL
-1+ agar 7gL
-1solid medium on carry out hairly root fast breeding cultivate, cultivating the propagation multiple of adding up afterwards hairly root for 25 days, be 1.86.
Above disclosed is only the preferred embodiments of the present invention, certainly can not limit with this interest field of the present invention, and the equivalent variations of therefore doing according to the present patent application the scope of the claims, still belongs to the scope that the present invention is contained.
Claims (10)
1. the genetic transforming method that the horseradish cotyledon of take is explant, is characterized in that comprising the steps:
Step 1, the activation culture of Agrobacterium bacterial classification: after Agrobacterium rhizogenes is rule on YEB solid medium, be placed under the temperature condition of 26～30 ℃ and carry out secretly cultivating after 24～36h, in picking list bacterium colony access YEB liquid nutrient medium, and, after 1～2 day, for explant, contaminate in 25～30 ℃ of shaking culture;
Step 2, the acquisition of horseradish aseptic seedling: wasabi seed is placed on Bechtop, first uses alcohol disinfecting 20～40s of 75%, again with 0.1% mercuric chloride sterilization 3～8min, after aseptic water washing 2～5 times, peel off kind of a skin and be seeded on substratum and cultivate, obtain horseradish aseptic seedling;
Step 3, the preculture of horseradish cotyledon: cut the cotyledon of horseradish aseptic seedling expansion as explant, be inoculated on pre-culture medium, and carry out preculture;
Step 4, the genetic transformation of horseradish cotyledon: choose horseradish cotyledon explant after preculture and put into the Agrobacterium bacterium liquid that step (1) activated and contaminate, after explant is fully contacted with Agrobacterium, and blot after the unnecessary bacterium liquid in explant surface with aseptic filter paper, proceed to again in common substratum, under the dark culture condition of 15～22 ℃, carry out the common cultivation of 1～3d; After being proceeded to subsequently in microbiotic water and soaking, then proceed to except carrying out degerming cultivation in bacterium culture medium, every 2～4d switching once except bacterium culture medium, until hairly root produces in a large number;
Step 5, the fast breeding of horseradish hairly root: the hairly root switching that cuts length and be 1.5～3cm is cultivated in hairly root fast breeding substratum.
2. a kind of genetic transforming method that horseradish cotyledon is explant of take according to claim 1, is characterized in that: the Agrobacterium adopting in step (1) is Agrobacterium rhizogenes A4 or R1000, contaminates the bacterium liquid OD of horseradish cotyledon explant
600=0.1～0.3.
3. a kind of genetic transforming method that horseradish cotyledon is explant of take according to claim 1, is characterized in that: in step (2), the wasabi seed after sterilization is peelled off to kind of skin and is seeded in MS+6-BA0.5～4mgL
-1+ NAA (or IAA) 0.5～2mgL
-1+ sucrose 20～50gL
-1+ agar 3.5～4.5gL
-1substratum on, and at 22～28 ℃, light intensity 1000～2000lx, cultivates under the condition of illumination every day 8～12h, obtains horseradish aseptic seedling.
4. a kind of genetic transforming method that horseradish cotyledon is explant of take according to claim 1, is characterized in that: in step (3), be to choose cotyledon that horseradish aseptic seedling just launched 1-4d as explant.
5. a kind of genetic transforming method that horseradish cotyledon is explant of take according to claim 1, is characterized in that: in step (3), pre-culture medium is MS+2,4-D1～2mgL
-1+ KT0.1～0.5mgL
-1+ VITAMIN B4 10～20mgL
-1+ sucrose 20～50gL
-1+ agar 5～7gL
-1.
6. a kind of genetic transforming method that horseradish cotyledon is explant of take according to claim 1, is characterized in that: in step (3), the preculture condition of horseradish cotyledon be 18～22 ℃, without carrying out the preculture of 1～3d under illumination condition.
7. a kind of genetic transforming method that horseradish cotyledon is explant of take according to claim 1, is characterized in that: in step (4), be to choose after preculture in horseradish cotyledon incision, to produce the explant expanding and put into the Agrobacterium bacterium liquid having activated and contaminate 1～3 minute.
8. a kind of genetic transforming method that horseradish cotyledon is explant of take according to claim 1, is characterized in that: the common substratum in step (4) is 1/2MS+ pyrocatechol 100～200mgL
-1+ sucrose 10～15gL
-1+ agar 4～5.5gL
-1; And under the dark culture condition of 15～22 ℃, carry out the common cultivation of 1～3d.
9. a kind of genetic transforming method that horseradish cotyledon is explant of take according to claim 1, is characterized in that: in step (4), the explant after cultivating is altogether proceeded to containing cefotaxime sodium 200～500mgL
-1microbiotic water in soak after 1～3 hour, then proceed to except bacterium culture medium be 1/2MS+IBA0.1～0.5mgL
-1+ agar 5～7gL
-1+ sucrose 15～30gL
-1+ cefotaxime sodium 200～400mgL
-1+ ceftriaxone sodium 200～400mgL
-1in carry out degerming cultivation.
10. a kind of genetic transforming method that horseradish cotyledon is explant of take according to claim 1, is characterized in that: in step (5), hairly root fast breeding substratum is 1/2MS+IBA0.1～0.5mgL
-1+ sucrose 10～20gL
-1+ cephamycin 100～300mgL
-1liquid nutrient medium on carry out hairly root fast breeding cultivate.
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| CN113584073A (en) * | 2021-09-14 | 2021-11-02 | 西南大学 | Method for establishing high-efficiency genetic transformation system of Arabidopsis thaliana |
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| CN111280066B (en) * | 2020-04-10 | 2022-09-13 | 成都大学 | Culture method of horseradish flower moss callus capable of effectively inhibiting browning |
| CN113584073A (en) * | 2021-09-14 | 2021-11-02 | 西南大学 | Method for establishing high-efficiency genetic transformation system of Arabidopsis thaliana |
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