CN102499087B - Isolated culture and plant regeneration method of silver chain - Google Patents
Isolated culture and plant regeneration method of silver chain Download PDFInfo
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Abstract
The invention discloses an isolated culture and plant regeneration method of silver chain, which is characterized in that: zygotic embryos with different embryo ages are adopted as an explant, mass spectrometry (MS) basic culture medium, 2-4-Morpholinoethanesulfonic acid (MES) 500mg/L, glutamine 250mg/L, caseinhydrolysate 500mg/L, rhodofix 0.1 to 1.0mg/L, Amino-6-benzyl adenine 0 to 1.0mg/L, cane sugar 30g/L and agar 6g/L are used as a body cell embryo induction culture medium, the silver chain somatic embryo can be obtained through the direct induction, and then the somatic embryo is cultured to be mature, sprouted and grown to obtain the regeneration plant. The induction rate of the somatic embryo in the method is high, and the average quantity of the somatic embryos of each explant reaches 5.4 to 10.8; and the sprouting rate and the transplanting survival rate of the somatic embryo are high, a great amount of excellent silver chain regenerated plants can be formed in a short term, and the method belongs to a simple and rapid technical system for factorized mass production of silver chain plants.
Description
Technical field
The present invention relates to a kind of method of Plant Tissue Breeding, the particularly method of locust tree cultured in vitro and plant regeneration belongs to the field of tissue culture of locust tree.
Background technology
Locust tree (Robinia pseudoacacia L.) claim again acacia, belong to pulse family (Leguminosae) Papillionoideae robinia (Robinia L.), deciduous tree, originate in the U.S., natural distributed is in A Bailaqi mountain range, eastern united states (Appalachian.Mt.) and Ao Sake mountain range (Ozank.Mt.), early in the twentieth century introduce China, enlarged rapidly after the introduction and planted, the Yellow River middle and lower reaches, Basin of Huaihe River, Haihe basin, the main timber forest of all provinces of THE LOWER YANGTZE VALLEY, firewood forest, soil and water conservation forest, sea wall and river levee shelter forest are only in Hebei, Henan, just there are 4,000,000,000 strains in 6 provinces and cities such as Shandong and Shanxi.Its fast growth is important fast-growing commerical tree species, and drought resistance, efficiency of preservation that its wood quality is hard, compression strength large, tool is stronger are strong.Locust tree root system length has a large amount of root nodules, can increase soil fertility by biological nitrogen fixation, plays a significant role at aspects such as keeping the ecological balance, raising stand quality.The blade of locust tree contains crude protein, is the feed stripped resource of high-quality cheapness.Pagoda Flower is nutritious, though short flower amount of florescence is considerable, except direct use, also can produces nectar, extract spices etc.
China starts from 20 century 70s to the breeding research of locust tree, has obtained so far a lot of achievements, but still can not satisfy the needs of production of forestry.The somatic embryo Regeneration Ways is that trees are bred Important Platform with genetic improvement, also is the regenerating system commonly used of realizing genetic transformation.Body embryo generation technique have many, the speed of breeding quantity soon, be not subjected to the affiliation restriction, in case but the general characteristics such as direct germination formation plantlet all of the somatic embryo that forms structural integrity are the plant regeneration systems of stability and high efficiency.In addition, somatic embryo is by unicellular origin, and development program is similar to zygotic embryo, can be used as the modular system of embryology research, and is also significant to the research of cell totipotency expression process and Cell Differentiation mechanism scheduling theory problem.Laine E and Dumet D successfully preserve pinus caribaea and oil palm embryo callus and have kept body embryo generation potentiality under ultralow temperature, still can continue after thawing to cultivate to form plant, and this may for saving that endangered plants provide.The cultured in vitro research of locust tree reached a climax after the eighties since late 1950s.
Up to the present, cultivate by forming, stem is cultivated, blade is cultivated by layer for locust tree, the cultivation of the ovary of not pollinating etc. obtained complete plant, but these researchs all concentrate on adventitious organogenesis, and the research by somatic embryo generation approach also seldom.For example, red gorgeous cotyledon take the locust tree mature seed is studied the embryo culture of locust tree body and evoking adventive bud as explant, also only has 36.7% but somatic embryo generation rate is the highest.
Studies show that in a large number the developmental stage of explant is to affect the key factor that somatic embryo occurs, immature zygotic embryos has significant advantage because its cell totipotency is high, dedifferentiation is easy when the inductor blast occurs.The most plants cultured in vitro generally adopts the immature zygotic embryos explant, but too young body embryonal induction rate tender or postmature zygotic embryo is unsatisfactory.In the growth course of whole zygotic embryo, only have short stage to have very strong body embryo generation potential.At present, the research zygotic embryo developmental stage is a lot of on the research of somatic embryo inducement impact, but with regard to locust tree, because the somatic embryo of locust tree is cultivated relatively difficulty, show as that body embryonal induction rate is low, Embryos quantity is many, body idioplasm amount is not high, restrict the degree of its genetic improvement, also hindered efficient breeding and the Rapid Popularization of choiceness.Up to now, the research of both at home and abroad somatic embryo of locust tree being cultivated is relatively less, the domestic relevant report that there is not yet, and external only Merkle et al. and Arrillaga et al. have delivered the result of study of this respect.Wherein, the people such as Merkle are from the 1st week after blooming, choosing immature zygotic embryos from 3 thorn Chinese scholartrees week about is explant, take the MS medium as minimal medium, studied explant embryo age to the impact of body embryonal induction, only observe somatic embryo from an immature zygotic embryos in rear 4 weeks of blooming and occur, the inductivity of its somatic embryo is low; The people such as Arrillaga have improved the inductivity of somatic embryo at the Research foundation of Merkle, take the FM medium as minimal medium, the bloom immature zygotic embryos in rear 1-4 week of employing is explant, carrying out somatic embryo inducement cultivates, result of the test shows that the explant body embryonal induction rate in the rear 2-3 week of blooming is the highest, only reaches 12%.Above-mentioned research all be the immature zygotic embryos of blooming rear 1-4 days be that explant is induced cultivation, its inductivity is low, the highest inductivity only is 12%, is difficult to satisfy the needs of locust tree breeding and breeding, therefore, it is imperative to set up locust tree active isomer embryo generation system.
Summary of the invention
Primary and foremost purpose of the present invention is to provide a kind of locust tree somatic embryo inducement rate height, the high and stable locust tree cultured in vitro Establish of regeneration method of germination rate for the problem that above-mentioned prior art exists.The method can form a large amount of good locust tree test tube strong sprouts within a short period of time, can carry out scale, batch production production, can be used for the research of locust tree transformation system, also can be used for molecular breeding research.
For achieving the above object, one aspect of the present invention provides a kind of locust tree propagation method, comprises following in sequence step:
1) gathers the locust tree pod;
2) the locust tree pod is carried out take out its zygotic embryo after surface sterilizing is processed, obtain aseptic zygotic embryo;
3) aseptic zygotic embryo is inoculated into carries out somatic embryo inducement in the somatic embryo inducement medium and cultivate, induce the generation proembryo;
4) proembryo is inoculated in the maturation of carrying out somatic embryo in the somatic embryo maturation medium and cultivates, obtain ripe somatic embryo;
5) somatic embryo of maturation is inoculated in sprouts cultivation on the germination medium, obtain body embryo seedling;
6) body embryo seedling is inoculated in carries out strong seedling culture on the strong seedling culture base, promote the growth of body embryo seedling, obtain test tube strong sprout;
7) test tube is carried out hardening, transplanting strong sprout, and get final product.
The explant embryo of locust tree is remarkable on the inductivity impact of callus age, and tender or overdone rataria all can not obtain desirable somatic embryo (being proembryo) and induce the result excessively.The present invention finds by a large amount of experiment, is in after blooming the locust tree pod in the 25th day to the 75th day this time period as explant, and its somatic embryo inducement effect is significantly improved than the pod in the section At All Other Times; Further experiment is found, collection is in locust tree bloom after pod in the 45th day to the 65th day this time period as the reproductive effect of explant further raising is arranged, employing is in locust tree bloom after pod in the 55th day to the 65th day this time period obtained further improving as the reproductive effect of explant, the 55th day locust tree pod had been obtained best reproductive effect as explant after employing was in and blooms: somatic embryo (proembryo) inductivity of immature zygotic embryos on described inducing culture after the collection locust tree blooms in the 55th day the pod all is higher than the explant in other period, its somatic embryo inducement rate is 92.4%, body embryo germination rate is 82.48-100%, all apparently higher than the explant of other period, thus the present invention most preferably gather be in bloom after the 55th day locust tree pod as explant.
Wherein, sterilization treatment step 2) comprises following in sequence step:
A) the locust tree pod that gathers is cleaned with sterile water after, behind alcohol-pickled pod, clean with aseptic water washing;
B) use HgCl
2Solution soaks pod, and is clean with aseptic water washing;
C) from pod, take out zygotic embryo after blotting the pod surface moisture, and get final product.
The concentration of volume percent of the alcohol particularly, steps A) is 75%; Soak time is 30s; Aseptic water washing 3-5 time.
Particularly, HgCl step B)
2The mass percent concentration of solution is 0.1%; Soak time is 3-7min, is preferably 5min; Aseptic water washing 5-6 time.
Wherein, step 3) medium of somatic embryo inducement described in is MS minimal medium+2-(N-morpholine) ethyl sulfonic acid (MES) 500mg/L+ glutamine 250mg/L+ caseinhydrolysate 500mg/L+ methyl α-naphthyl acetate 0.1-1.0mg/L+6-benzyl aminoadenine 0-1.0mg/L+ sucrose 30g/L+ agar 6g/L, and the pH value is 5.8.
Particularly, the somatic embryo inducement medium optimization is: MS minimal medium+MES 500mg/L+ glutamine 250mg/L+ caseinhydrolysate 500mg/L+ methyl α-naphthyl acetate 0.1-1.0mg/L+6-benzyl aminoadenine 0.1-1.0mg/L+ sucrose 30g/L+ agar 6g/L, and the pH value is 5.8; More preferably: MS minimal medium+MES 500mg/L+ glutamine 250mg/L+ caseinhydrolysate 500mg/L+ methyl α-naphthyl acetate 0.5mg/L+6-benzyl aminoadenine 0.5mg/L+ sucrose 30g/L+ agar 6g/L, the pH value is 5.8.
The immature zygotic embryos integral body that particularly, will obtain from the pod in the rear 4-11 week of blooming is inoculated into carries out described somatic embryo inducement cultivation in the described somatic embryo inducement medium.
Particularly, somatic embryo inducement step 3) is cultivated and carried out under the following conditions: dark condition, cultivation temperature are 26 ± 2 ℃.
Especially, relative moisture is 70-75% in the somatic embryo inducement incubation.
Wherein, step 4) the somatic embryo maturation medium described in is MS minimal medium+caseinhydrolysate 500mg/L+ sucrose 30g/L+ agar 6g/L, and the pH value is 5.8; Step 5) germination medium described in is MS minimal medium+sucrose 30g/L+ agar 6g/L, and the pH value is 5.8; Step 6) the strong seedling culture base described in is MS minimal medium+IBA 0.2mg/L+ sucrose 30g/L+ agar 6g/L, and the pH value is 5.8.
Particularly, the ripe cultivation of somatic embryo step 4) carried out under the following conditions: cultivation temperature is 26 ± 2 ℃, and intensity of illumination is 1500-2000lux, and periodicity of illumination is 14-16 hour illumination/8-10 hour dark.
Especially, relative moisture is 70-75% in the ripe incubation of somatic embryo.
Particularly, sprout step 5) to cultivate and carry out under the following conditions: cultivation temperature is 26 ± 2 ℃, and intensity of illumination is 1500-2000lux, and periodicity of illumination is 14-16 hour illumination/8-10 hour dark.
Especially, relative moisture is 70-75% in the sprouting incubation.
Particularly, strong seedling culture is carried out under the following conditions step 6): cultivation temperature is 26 ± 2 ℃, and intensity of illumination is 1500-2000lux, and periodicity of illumination is 14-16 hour illumination/8-10 hour dark.
Especially, relative moisture is 70-75% in the strong seedling culture process.
Wherein, hardening, transplanting step 7) comprise following in sequence step: open the bottle cap of blake bottle, hardening is 1 day in transplanting the chamber; Then test tube is taken out strong sprout, clean the residual agar medium of test tube root in strong sprout with running water, be transplanted in the locust tree soilless culture substrate and transplant cultivation.
Particularly, also comprise with transplanting cultivate 5-6 after week plant be colonizated in again the land for growing field crops.
Particularly, also comprise test tube is positioned over to cultivate under the natural lighting intensity of shading 50% strong sprout and open again the blake bottle bottle cap after 5-10 days.
Particularly, described locust tree soilless culture substrate is comprised of perlite, vermiculite, and wherein perlite is 1: 1 with the ratio of the volume of vermiculite.
Particularly, transplanting cultivation is under the natural lighting condition, cultivates 5-6 week
Especially, transplanting cultivation temperature is that 25 ± 5 ℃, relative moisture are 56-90%.
The propagation method of locust tree of the present invention has the following advantages:
1, the present invention utilizes the locust tree immature zygotic embryos to carry out Vitro Quick Reproduction, the minimal medium that each cultivation stage adopts be the MS minimal medium, has the inorganic salt concentration height, particularly the content of nitrate, potassium and ammonium is high, can required mineral nutrition is provided and promote somatic embryo inducement, maturation and germination and growth for tissue growth.
2, the locust tree reproductive efficiency is high in the propagation method of the present invention, adding plant growth regulator in the medium is beneficial to somatic embryo and directly induces, breeds from explant, maturation and sprouting, composition of nutritive substance is reasonable in the locust tree medium that uses among the present invention, consumption and proportioning are suitable, cultivate result's repeatability height, and regenerating system is stable, the inductivity 63.10-92.40% of somatic embryo, each explant average body blast number reach 5.4-10.8.
3, the locust tree regeneration plant robust growth of the inventive method cultivation, reproduction coefficient height, the somatic embryo germination rate is high, reaches 72-100%; Transplanting survival rate is high, reaches 100%, is the simple, fast technical system of batch production large-scale production locust tree plant.
Description of drawings
Fig. 1 is that the somatic embryo inducement of locust tree immature zygotic embryos is cultivated the proembryo that obtains;
Fig. 2 is the ripe mature somatic embryo that obtains of cultivating of locust tree proembryo;
Fig. 3 is the microscopic photo of locust tree mature somatic embryo thin layer section;
Fig. 4 is that the locust tree mature somatic embryo is sprouted the body embryo seedling that cultivation obtains;
Fig. 5 is the test tube strong sprout that locust tree body embryo seedling strong seedling culture obtains;
Fig. 6 is the BLACK LOCUST SEEDLINGS of transplanting in container.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not consisted of any restriction.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can make amendment or replace the details of technical solution of the present invention and form, but these modifications and replacing all fall within the scope of protection of the present invention.
Embodiment 1
One, test material
1, the present invention with Yanqing, Beijing rice man fort nursery well-grown dliploid locust tree seedling colony for the resource of drawing materials, choose 10 well-growns, dry type is more straight, the elite stand that ripening rate is high (interval between each strain is greater than 50 meters), between in June, 2010 to September, the zygotic embryo of the locust tree different developmental phases in 4-12 week was as the explant test material after collection was bloomed, every interval gathered once in 10 days, 500 pods of each collection, namely gather and bloom rear 25,35,45,55,65,75 days pod, 500 pods of each collection, the pod that will gather same day low temperature in ice chest is preserved and to be taken back the laboratory, and it is for subsequent use to be placed on afterwards in 4 ℃ the refrigerator refrigeration.
2, plant growth regulator
Employed plant growth regulating substance adopts domestic methyl α-naphthyl acetate (NAA), 6-benzyl aminoadenine (6-BA) among the present invention.
3, the preparation of medium:
(1) composition or the compound method of " MS minimal medium ";
Table 1MS medium (Murashige and Skoog, 1962)
After above-mentioned MS medium mother liquor prepared, it was stand-by to be stored in 4 ℃ of refrigerators.Quantity according to the configuration medium, the required agar of weighing and sucrose, be poured into and want to join in the sterile water of culture volume 3/4, order adds required macroelement mother liquor, micro-mother liquor and organic principle mother liquor, every adding is a kind of all fully stirs, add at last water and be settled to the medium final volume, with pH meter test media acid-base value, with the NaOH of 1mol/L or the HCl of 1mol/L the pH value is adjusted to 5.8.
(2) somatic embryo inducement medium: the MS minimal medium adds NAA 0.1-1.0mg/L, 6-BA0-1.0mg/L, MES 500mg/L, glutamine 250mg/L, caseinhydrolysate 500mg/L, sucrose 30g/L, agar 6g/L, regulating the pH value is 5.8, and medium was 121 ℃ of lower constant temperature sterilizations 15 minutes.
(3) maturation medium: the MS minimal medium adds caseinhydrolysate 500mg/L, sucrose 30g/L, agar 6g/L, and regulating the pH value is 5.8.Medium was 121 ℃ of lower constant temperature sterilizations 15 minutes.
(4) germination medium: the MS minimal medium adds sucrose 30g/L, agar 6g/L, and regulating the pH value is 5.8, and medium was 121 ℃ of lower constant temperature sterilizations 15 minutes.
(5) strong seedling culture base: the MS minimal medium adds IBA 0.2mg/L, sucrose 30g/L, agar 6g/L, and regulating the pH value is 5.8, and medium was 121 ℃ of lower constant temperature sterilizations 15 minutes.
4, condition of culture
The condition of culture that somatic embryo inducement is cultivated: closed tissue culture room, under the dark condition, cultivation temperature is 26 ± 2 ℃.
The ripe cultivation of somatic embryo, mature somatic embryo are sprouted the condition of culture of cultivation, body embryo seedling strong seedling culture: cultivation temperature is 26 ± 2 ℃, and intensity of illumination is 1500-2000lux, and periodicity of illumination is 14-16 hour illumination/8-10 hour dark.
Two, test method
1, the sterilization of explant
To gather respectively bloom after 25,35,45,55,65,75 days pod wash, be behind 75% the alcohol-pickled pod 30s, with aseptic water washing several (3-5 time) at the superclean bench concentration of volume percent; Then be 0.1% HgCl with mass percent concentration
2Solution soaks pod 3-7min (being preferably 5min), with aseptic water washing several (5-6 time); Then pod is placed on and from pod, takes out immature seed after blotting surface moisture on the filter paper of aseptic drying, i.e. Immature zygotic embryo, the immature zygotic embryos of 25,35,45,55,65,75 days surface sterilizing is for subsequent use after obtaining blooming.
2, somatic embryo inducement is cultivated
After in superclean bench, will blooming 25,35,45,55,65, the immature zygotic embryos integral body of 75 days and surface sterilizing is inoculated in the somatic embryo inducement medium as explant, under dark condition, carry out the cultivation of inducing of locust tree somatic embryo, every 3-4 week subculture once, the immature zygotic embryos that is about to sterilization is cultivated the afterwards taking-up of 3-4 week, place another fresh somatic embryo inducement medium to proceed the cultivation of inducing of somatic embryo, to keep medium to contain sufficient nutriment and moisture, wherein, used 6-BA is 0.5mg/L in the somatic embryo inducement medium, NAA is 0.5mg/L, inducing cultivation temperature is (26 ± 2) ℃, and relative moisture is 70-75% in the incubation, induces the edges of cultivating after 6 weeks at explant, can observe embryoid projection not of uniform size, be yellow green, polydispersion distributes, after subculture is cultivated 2 times, obtain proembryo (such as Fig. 1), statistics is induced and is obtained former embryo explants number, calculates body embryonal induction rate, and the result is as shown in table 2.
In the somatic embryo inducement incubation, the each processing inoculated 10 of immature zygotic embryos, reprocessing 3 times.
The explant number of somatic embryo inducement rate (%)=induce somatic embryo (proembryo)/inoculation explant sum * 100%.
3, the maturation of somatic embryo is cultivated
Proembryo integral body is transferred to carries out on the somatic embryo maturation medium that somatic embryo is ripe to be cultivated, wherein condition of culture is as follows: 26 ± 2 ℃ of cultivation temperature, photoperiod is that 16h illumination/8h is dark, intensity of illumination is 1500-2000Lux, relative moisture is 70-75%, and per 3 all subcultures once in the ripe incubation of somatic embryo.After cultivating for 4 weeks, proembryo is individual to be increased, and it is green that color turns gradually, forms the organism that is similar to zygotic embryo, obtains mature somatic embryo (as shown in Figure 2).
Fix with the agarose embedding inducing the somatic embryo that obtains, automatically shake the microtome section with come card UC6 type, thickness is 100 microns, then with the dyeing of rhodamine dyeing liquor, at Olympus optical microphotograph Microscopic observation, microscopic result such as Fig. 3, the microscopy result shows that somatic embryo grown cotyledon period, the somatic embryo of this moment has root meristematic tissue and stem meristematic tissue, shows obvious bipolarity.
4, the sprouting of somatic embryo is cultivated
The somatic embryo of maturation is separated from the explant piece, be transferred on the somatic embryo germination medium, place under 26 ± 2 ℃ of conditions, be that 16h illumination/8h is dark in the photoperiod, intensity of illumination is to cultivate under the 1500-2000Lux condition, the relative moisture of sprouting in the incubation is 70-75%, the mature somatic embryo hypocotyl elongation, Fast Growth, cultivated for 4 weeks after, the cotyledon on the top of mature somatic embryo launches, the other end differentiates obvious root growth end, organizator embryo seedling (such as Fig. 4), and statistics is sprouted and is obtained body embryo seedling number, calculate somatic embryo germination rate (body embryo germination rate), result such as table 2.
Sprout in the incubation at somatic embryo, process 10 of inoculum blasts, reprocessing 3 times at every turn.
The regenerate embryo seedling number that body embryo germination rate (%)=sprouting obtains/inoculum blast sum * 100%
5, the strong seedling culture of body embryo seedling
With body embryo seedling global transfer in the strong seedling culture base, place under 26 ± 2 ℃ of conditions, be that 16h illumination/8h is dark in the photoperiod, intensity of illumination is to carry out strong seedling culture under the 1500-2000Lux condition, and the relative moisture in the strong seedling culture process is 70-75%, per 4 all subcultures are once in the strong seedling culture process for body embryo seedling, the root of body embryo seedling extends gradually, forms taproot and lateral root, and plumule is grown and is stem and leaf, after cultivating for 6 weeks, obtain test tube strong sprout (such as Fig. 5).
6, hardening, transplanting, the field planting in test tube strong sprout
Test tube strong seedling culture bottle is placed in the seeding room of shading condition (shading rate is 50%) and cultivated 5-10 days, then open the blake bottle bottle cap, hardening was cultivated after 1 day in transplanting the chamber, take out plant, clean the residual agar medium of test tube root in strong sprout with running water, be transplanted to locust tree soilless culture substrate (perlite, vermiculite, perlite is 1: 1 with the ratio of the volume of vermiculite) in carry out container and cultivate transplanting the chamber, transplanting cultivation temperature is 25 ± 5 ℃, relative moisture is 56-90%, after natral light cahure 7-10 days, the statistics transplanting survival rate reaches 100%, as shown in Figure 6.Transplant 5-6 and after week BLACK LOCUST SEEDLINGS is colonizated in the land for growing field crops again.
Embodiment 2
In the somatic embryo inducement incubation, the used 6-BA of somatic embryo inducement medium is 1.0mg/L, and NAA is outside the 1.0mg/L, and all the other are identical with embodiment 1, and data results is as shown in table 2.
Table 2 somatic embryo inducement cultivation results
Embodiment 3
In the somatic embryo inducement incubation, the used 6-BA of somatic embryo inducement medium is 0.1mg/L, and NAA is outside the 0.1mg/L, and all the other are identical with embodiment 1, and data results is as shown in table 3.
Embodiment 4
In the somatic embryo inducement incubation, the used 6-BA of somatic embryo inducement medium is 0.1mg/L, and NAA is outside the 0.5mg/L, and all the other are identical with embodiment 1, and data results is as shown in table 3.
Table 3 somatic embryo inducement cultivation results
Embodiment 5
In the somatic embryo inducement incubation, the used 6-BA of somatic embryo inducement medium is 0mg/L, and NAA is outside the 0.1mg/L, and all the other are identical with embodiment 1, and data results is as shown in table 4.
Embodiment 6
In the somatic embryo inducement incubation, the used 6-BA of somatic embryo inducement medium is 0.5mg/L, and NAA is outside the 1.0mg/L, and all the other are identical with embodiment 1, and data results is as shown in table 4.
Table 4 somatic embryo inducement cultivation results
Have table 2,3,4 result of the test to draw:
1, explant embryo age, excessively tender (early than blooming rear 45 days) or the rataria of overdone (be later than and bloom rear 65 days) all can not obtain desirable body embryonal induction result on the direct inductivity impact of somatic embryo significantly (p＜0.05).The somatic embryo generation rate trend under but the concentration combination of each growth regulator is induced is roughly the same, and the body embryo formation frequency of immature zygotic embryos under each growth regulating agent concentration of the collection in rear 55 days of blooming all is higher than the explant of other developmental stage.
2, in the inventive method take MS as minimal medium, to add NAA 0.5mg/L, 6-BA 0.5mg/L, MES500mg/L, glutamine 250mg/L, caseinhydrolysate 500mg/L, sucrose 30g/L, agar 6g/L, the pH value is that 5.8 medium is the somatic embryo inducement medium, the somatic embryo inducement rate is high, on the average body embryonal induction number impact remarkable (p＜0.05) of single explant.
The explant body embryonal induction rate of 3, blooming rear 55 days among the present invention is 92.40 ± 0.12%, the average body embryo number of each explant is 10.80 ± 0.17, all apparently higher than the explant in other periods, take the rear best period of drawing materials as immature zygotic embryos in 55 days of blooming.
4, the explant embryo is remarkable on the sprouting yield impact of somatic embryo age in the inventive method, the sprouting yield of somatic embryo is high, reach 72-100%, the sprouting yield of the immature zygotic embryos that gathered in rear 55 days of blooming all is higher than the explant of other developmental stage, and somatic embryo is sprouted yield and reached 82-100%.
Claims (8)
1. the method for the cultured in vitro of a locust tree and plant regeneration comprises following in sequence step:
1) gather the locust tree pod, wherein, described locust tree pod is the locust tree pod in the 25th day to the 75th day this time period after being in locust tree and blooming;
2) the locust tree pod is carried out take out its zygotic embryo behind the surface sterilizing, obtain aseptic zygotic embryo;
3) aseptic zygotic embryo is inoculated into carries out somatic embryo inducement in the somatic embryo inducement medium and cultivate, induce the generation proembryo, wherein, described somatic embryo inducement medium is MS minimal medium+2-N-morpholine ethyl sulfonic acid 500mg/L+ glutamine 250mg/L+ caseinhydrolysate 500mg/L+ methyl α-naphthyl acetate 0.1-1.0mg/L+6-benzyl aminoadenine 0-1.0mg/L+ sucrose 30g/L+ agar 6g/L, and the pH value is 5.8;
4) proembryo being inoculated in the maturation of carrying out somatic embryo in the somatic embryo maturation medium cultivates, obtain mature somatic embryo, wherein, described somatic embryo maturation medium is MS minimal medium+caseinhydrolysate 500mg/L+ sucrose 30g/L+ agar 6g/L, and the pH value is 5.8;
5) somatic embryo of maturation is inoculated in sprouts cultivation on the germination medium, obtain body embryo seedling, wherein, described germination medium is MS minimal medium+sucrose 30g/L+ agar 6g/L, and the pH value is 5.8;
6) body embryo seedling is inoculated in carries out strong seedling culture on the strong seedling culture base, promote the growth of body embryo seedling, obtain test tube strong sprout, wherein, described strong seedling culture base is MS minimal medium+IBA 0.2mg/L+ sucrose 30g/L+ agar 6g/L, and the pH value is 5.8;
7) test tube is carried out hardening, transplanting strong sprout, and get final product.
2. the method for claim 1 is characterized in that the locust tree pod described in the step 1) is the locust tree pod in the 45th day to the 65th day this time period after being in locust tree and blooming.
3. the method for claim 1 is characterized in that the locust tree pod described in the step 1) is the locust tree pod in the 55th day to the 65th day this time period after being in locust tree and blooming.
4. the method for claim 1 is characterized in that the locust tree pod described in the step 1) is to be in the locust tree pod that locust tree bloomed rear the 55th day.
5. the method for claim 1, it is characterized in that somatic embryo inducement described in the step 3) is cultivated carries out under the following conditions: under the dark condition, cultivation temperature is 26 ± 2 ℃.
6. the method for claim 1, it is characterized in that ripe cultivation of somatic embryo described in the step 4) carry out under the following conditions: cultivation temperature is 26 ± 2 ℃, and intensity of illumination is 1500-2000lux, and periodicity of illumination is 14-16 hour illumination/8-10 hour dark.
7. the method for claim 1, it is characterized in that sprouting described in the step 5) to cultivate carrying out under the following conditions: cultivation temperature is 26 ± 2 ℃, and intensity of illumination is 1500-2000lux, and periodicity of illumination is 14-16 hour illumination/8-10 hour dark.
8. the method for claim 1 is characterized in that:
Step 2) sterilization treatment described in comprises following in sequence step:
A) the locust tree pod that gathers is cleaned with sterile water after, behind alcohol-pickled pod, clean with aseptic water washing;
B) use HgCl
2Solution soaks pod, and is clean with aseptic water washing;
C) blot and from pod, take out immature seed behind the pod surface moisture, and get final product;
Acclimatization and transplants described in the step 7) comprises following in sequence step: open the bottle cap of blake bottle, hardening is 1 day in transplanting the chamber; Then test tube is taken out strong sprout, clean the residual agar medium of test tube root in strong sprout with running water, be transplanted in the locust tree soilless culture substrate and transplant cultivation.
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| CN104686327A (en) * | 2015-02-21 | 2015-06-10 | 杨业云 | Method for establishing tissue culture rapid propagation system of single-leaf robinia. pseudoacacia |
| CN106613971B (en) * | 2016-12-08 | 2019-08-16 | 上海杉一植物科技有限公司 | A kind of culture of rootage method of the fast-growing without locust tree tissue-cultured seedling |
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| Title |
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| "Advances in somatic embryogenesis and plant production of black locust (Robinia pseudoacacia L.)";I. Arrillage等;《Plant Cell Reports》;19941231;第13卷;第171-175页 * |
| "植物激素对刺槐器官发生及其植株再生影响的研究";红艳等;《内蒙古农业大学学报》;20070630;第28卷(第2期);第55-59页 * |
| "饲料型四倍体刺槐幼胚离体培养挽救技术研究";贺佳玉等;《河北林果研究》;20080630;第23卷(第2期);第113-117页 * |
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| 红艳等."植物激素对刺槐器官发生及其植株再生影响的研究".《内蒙古农业大学学报》.2007,第28卷(第2期),第55-59页. |
| 贺佳玉等."饲料型四倍体刺槐幼胚离体培养挽救技术研究".《河北林果研究》.2008,第23卷(第2期),第113-117页. |
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