CN103004589B - Tissue culture and rapid propagation method of agrimony - Google Patents
Tissue culture and rapid propagation method of agrimony Download PDFInfo
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- CN103004589B CN103004589B CN201210505869.2A CN201210505869A CN103004589B CN 103004589 B CN103004589 B CN 103004589B CN 201210505869 A CN201210505869 A CN 201210505869A CN 103004589 B CN103004589 B CN 103004589B
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Abstract
The invention relates to a tissue culture and rapid propagation method of plants and particularly relates to a tissue culture and rapid propagation method of tulipa edulis. The method comprises the following steps that firstly, explants are disinfected, wherein the large leaflets of pinnately compound leaves of the fully-developed tulipa edulis are washed and disinfected and then are cut into small pieces of 1 to 3cm<2>, and then the small pieces with upward front surfaces are horizontally inoculated into an MS (Murashige and Skoog) culture medium with different plant growth regulating substances; secondly, adventitious bud is induced, wherein the explants are inoculated into the MS culture medium; and finally, the adventitious buds of the leaves, which are light green, grow vigorously and are 2cm to 3cm high, are cut, the adventitious buds are inserted into a rooting medium, the adventitious buds are taken out from the medium after 20-30 hours, washing the medium adhered to the roots of the adventitious buds are washed, the adventitious buds are transplanted into earth, and a beaker is removed after young leaves grow. The tissue culture and rapid propagation method has the advantages that the rooting rate and the rotting number are increased, the length and thickness of roots are moderate, and the survival rate of transplanting is greatly increased.
Description
Technical field
The present invention relates to Plant Tissue Breeding and method for quickly breeding, the tissue that relates in particular to a kind of Agrimony is cultivated and method for quickly breeding.
Background technology
Agrimony (
agrimonia pilosa) belong to herbaceos perennial for the rose family (Rosaceae) Agrimony, be a kind of important medicinal plant, its acrial part, hibernaculum and root with short and small rhizome all can be used as medicine.Wherein, acrial part is used as medicine and claims hairyvein agrimony, different name fortune silene herb with root, wolf's fang grass, goldentop fossilia dentis mastodi, hairyvein agrimony, Agrimony, Huanglong's tail, triflorous ainsliaea leaf, dry foot mattress etc., property is put down, bitter, puckery, return lung, liver, the spleen channel, Bearberry Extract hemostasis, anti-inflammtory anti-dysentery, desinsection and antitumor effect, the clinical infectious food poisoning of Halophiles, trichomonas vaginitis and the meniere's syndrome of being used for the treatment of obtains good effect; Be used as medicine and claim gemma agrimoninae with the hibernaculum of short and small rhizome, different name wolf's fang, wolf tooth, wolf's fang, wolf's fang grass roots bud, hairyvein agrinonia rhizome etc., it is cool in nature, bitter, puckery, effect of tool expelling parasite and removing toxicity for detumescence, cures mainly tapeworm disease, trichomoniasis, sore mange, furuncle and dysentery.Clinical treatment tapeworm disease has good effect; A root hairyvein agrimonia root that is used as medicine, different name frozen ground wind, it is warm in nature, and taste is pungent, puckery, and effect of tool removing toxic substances and expelling parasite, cures mainly the diseases such as dysentery, sore, pyogenic infections, malaria, tapeworm disease and amenorrhoea.In the last few years, the chemical composition analysis to Agrimony, extraction separation, assay method and effect had had more deep research.The breeding of current imperial bud adopts seed and root division mode to carry out, and these two kinds of methods exist floods the shortcoming that reproduction coefficient is low, the breeding cycle is long, therefore sets up the tissue-culturing rapid propagation system of Agrimony, is to realize the another effective way that its industrialization is produced.
Report (" Plant Physiology Communications " 04 phase in 1986 that existing Agrimony tissue is cultivated, the tissue of Agrimony is cultivated, Wu Meifen, Chen Weidong): the adventitious organogenesis taking hypocotyl, cotyledon and the blade of Agrimony seedling as explant, blade and hypocotyl need be through 2, in the 4-D evoked callus stage, cotyledon can differentiate indefinite bud after MS+6-BA 2.0 mg/L NAA 0.2 mg/L evoked callus.
Summary of the invention
In order to solve above-mentioned technical problem, the tissue that the object of this invention is to provide a kind of Agrimony is cultivated and method for quickly breeding, the tissue that adopts the method to carry out Agrimony is cultivated and Fast-propagation, improve rooting rate and the number of taking root, and length and the rugosity of root are moderate, greatly improve the survival rate of transplanting.
In order to realize above-mentioned object, the present invention has adopted following technical scheme:
The tissue of Agrimony is cultivated and method for quickly breeding, and the method comprises the following steps:
1) sterilization of explant:
Get the larger leaflet in firm fully-developed Agrimony winglike compound leaf, add washing agent and carefully scrub, then rinse with flowing water, in super-clean bench, blade is proceeded in alcoholic solution and soaked, after proceed to soaking disinfection in thimerosal, with aseptic water washing, then blade is cut into 1 ~ 3 cm
2fritter, be inoculated into and add on the MS medium of different plant growth regulating substances with heads horizontal disposing way;
2) adventitious bud inducing
The composition of the plant growth regulating substance adding in the MS medium of explant access comprises:
6-BA 0.5~5.0 mg/L
TDZ 0.1~1.0 mg/L；
First secretly cultivate, temperature is 25 ± 1 DEG C, and condition of culture is light/dark hour than being 12 ~ 16:8 ~ 12, intensity of illumination 30 ~ 40 μ mol/ms;
3) cut that blade is light green, the indefinite bud vigorous and that 2 ~ 3 cm are high of growing, be inserted in root media, described root media comprises:
Minimal medium (1/4 ~ 1) MS
NAA 0.05~0.5 mg/L；
When hardening, crack cultivation bottle cap carries out hardening, and the medium that after 20 ~ 30h, taking-up clean root adhere to moves in soil, and the initial stage will be carried out moist keeping measures, and In Shade, treats that young leaves grows, and can remove beaker.
As preferably, above-mentioned step 1) thimerosal is the 0.2%HgCl that drips a 1 mol/L NaOH
2solution.Be 10 ~ 11 min as preferred again, above-mentioned disinfecting time.
As preferably, above-mentioned step 2) in the composition of the plant growth regulating substance that adds comprise:
6-BA 1.0~4.0 mg/L
TDZ 0.1~0.8mg/L。
As most preferably, above-mentioned step 2) in the plant growth regulating substance that adds select TDZ 0.3 mg/L+6-BA 2.0 mg/L.
As preferably, above-mentioned step 2) composition of plant growth regulating substance also comprises IAA, NAA or both combinations, and the concentration of IAA is 0 ~ 2.0 mg/L, and the concentration of NAA is 0 ~ 0.5 mg/L.
As preferred, above-mentioned again step 2) composition of plant growth regulating substance also comprises IAA, the concentration of IAA is 0.5 ~ 1.5 mg/L.
As preferably, in above-mentioned step 3) root media, NAA is 0.08 ~ 0.3mg/L.
As most preferably, above-mentioned step 3) root media is 1/4MS+NAA 0.1 mg/L.
The present invention is owing to having adopted above-mentioned technical scheme, and what adopt that the method carries out Agrimony has improved rooting rate and the number of taking root with Fast-propagation, and the length of root and rugosity moderate, greatly improved the survival rate of transplanting.The method has been set up the tissue-culturing rapid propagation system of Agrimony, is to realize the effective way that its industrialization is produced.
Embodiment
Material source: Agrimony is picked up from White Cloud Mountain, Lishui, transplants and plants to solarium of the China Measures Institute.
Explant material: the blade of Agrimony.
1. the sterilization of explant: get the larger leaflet in firm fully-developed Agrimony winglike compound leaf, adding appropriate washing agent carefully scrubs, rinse after 0.5 ~ 1.0 h with flowing water again, in super-clean bench, blade is proceeded in 70% alcoholic solution and soaks 30 s, after proceed to the 0.2%HgCl that has dripped 11 mol/L NaOH
2the soaking disinfection some time in solution, use aseptic water washing 4 ~ 5 times, then blade is cut into 1 ~ 3 cm
2fritter, be inoculated into and add on the MS medium of different plant growth regulating substances with heads horizontal disposing way.
1.1 results: sterilization 5 ~ 6 min reach optimum efficiency, and the pollution rate of explant and lethality are all lower than 10%.
2. adventitious bud inducing preliminary experiment
The composition of the plant growth regulating substance adding in the MS medium of explant access is respectively:
1) 2,4-D 2.0 mg/L +6-BA 0.5 mg/L；
2) 6-BA 2.0 mg/L +NAA 0.1 mg/L；
3) TDZ 0.3 mg/L
-1+NAA 0.1 mg/L；
4) TDZ 0.05 mg/L +2,4-D 2.0 mg/L；
5) TDZ 0.3 mg/L +6-BA 2.0 mg/L。
Repeat 3 times.In all medium, sucrose concentration is 3%, and agar concentration is 0.7%, pH 5.8-6.0, lower same.First secretly cultivate, temperature is (25 ± 1) DEG C, and condition of culture is that 14h light/10h is dark, and intensity of illumination 30 ~ 40 μ mol/ms are lower same.Observe at ordinary times explant growth situation record, extract 10 bottles out and add up at random in the time of 30 d, and filter out preferably plant growth regulating substance and combine to be optimized.
2.1 results: add 2, in the medium (1 and No. 4 medium) of 4-D, do not induce indefinite bud, and in all the other 3 kinds of medium, all induce indefinite bud, but the time that indefinite bud occurs differs, to add TDZ 0.3 mg/L+6-BA 2.0 mg/L (No. 5 medium) the earliest, the earliest can 12 d, inductivity is the highest (65.6%) also, add 6-BA 2.0 mg/L+NAA 0.1 mg/L (No. 2 medium) the latest, earliest time is 20 d, inductivity is minimum (12.2%) also, therefore select TDZ and 6-BA as candidate plant growth regulating substance in the screening of best adventitious bud inducing.Simultaneously, in incubation, observe, in the medium of interpolation 2,4-D, only observe less more tissue, and in the medium of generation indefinite bud, after finding certain hour, its part leaf dish base portion expands, edge perk, and paddle cutout place has the green matter of similar callus to form, and base portion is more than end, but be carefully distinguished as budlet point, and more obvious along with the increase of incubation time, occur part that indefinite bud is maximum be leave handle place.
3. the optimization of the plant growth regulating substance of adventitious bud inducing combination
TDZ (0.1,0.3 and 1.0 mg/L) and 6-BA (0.5, the 2.0 and 5.0 mg/L) combination of 3 gradients are respectively set, and preparation MS cultivates, and explant is inoculated thereon, and 30 d extract 10 bottles out at random and add up.
The rising of 3.1 results: TDZ and 6-BA concentration, adventitious bud induction frequency and induction number show zooming trend, but decline to some extent in the time of high concentration, and each inductivity of processing is between 44.1 ~ 8.9%, and the indefinite bud quantity inducing is between 3.3 ~ 7.7.The synergistic effect of two plant growth regulating substances is also very obvious, especially when low concentration, the raising of a plant growth regulating substance concentration can improve inductivity and induction number fast, and adventitious bud induction frequency reaches as high as 90% left and right, and the indefinite bud inducing also has 8 left and right.But the TDZ of high concentration and 6-BA have brought vitrified problem, both also have synergistic effect, the TDZ of maximum concentration and 6-BA combination, and its vitrifying is the most serious.The vitrifying degree of moderate TDZ and 6-BA is lower, and adventitious bud induction frequency is higher with induction number, and especially good with being combined as of TDZ 0.3 mg/L+6-BA 2.0 mg/L, inductivity can arrive 85.6%, and indefinite bud quantity can reach 7.4, and well-grown.
4. different growth regulatory substances combine the impact on proliferate
Though MS+TDZ 0.3 mg/L+6-BA 2.0 mg/L can induce indefinite bud, though find that in subculture indefinite bud has propagation, poor growth, when 30 d seldom higher than 2 cm, the experiment that is difficult to take root, therefore in proliferate is cultivated, more additionally add growth hormone IAA and NAA to screen.By the indefinite bud clump inducing in MS+TDZ 0.3 mg/L+6-BA 2.0 mg/L medium, be cut into the fritter of 2 ~ 3 strains, variable concentrations IAA (0,0.2,0.5,1.0 and 2.0 mg/L) or NAA (0,0.05,0.1,0.2 and 0.5 mgL are additionally added in inoculation again
-1) Optimal Medium in.30 d extract 10 bottles out at random and add up.
4.1 results: along with the rising of IAA concentration, the rate of increase of indefinite bud improves, and growth is also accelerated simultaneously, and reach stable at 1 mg/L, the rate of increase can be elevated to 4.39 from 4.03, but NAA is all little on the impact of the rate of increase and indefinite bud growth, between 4.09 ~ 4.14.
5. the culture of rootage of regeneration plant and transplanting
Cut that blade is light green, the indefinite bud vigorous and that 2 ~ 3 cm are high of growing, be inserted in root media, minimal medium (MS, 1/2MS and 1/4MS), NAA (0,0.1 and 0.5 mg/L) and the inositol (0%, 50%, 100%) of 3 gradients are respectively set, when 30 d, extract 10 bottles out at random and add up rooting rate.When hardening, can carry out hardening by crack cultivation bottle cap, the medium that after 1 d, taking-up clean root adhere to, moves in soil, and soil is little silt, and the initial stage will be carried out moist keeping measures, and In Shade, treats that young leaves grows, and can remove beaker, adds up transplanting survival rate simultaneously.
5.1 results: Agrimony takes root easily, each rooting rate of processing is between 88 ~ 98%.Along with MS concentration reduces, rooting rate improves, and radical amount increases, elongated, attenuates; And along with the increase of NAA concentration, taking root takes the lead in raising declines again, the quantity of root first increases and reduces, and the length of root also shows and first increases the trend reducing afterwards, but the rugosity of root shows the trend of increase; But the concentration of inositol is little for culture of rootage impact.In addition, observe and find, each rootage duration of processing is close, and with 1/4MS+NAA 0.1 mg/L, (6 d), and with MS, (8 d) the latest the earliest.But on transplanting survival rate, have any different, when high concentration (0.5 mg/L) NAA cultivates, although the quantity of root increases, but elongated, shorten, the lignification of these roots is obvious, it is thick and hard that root becomes, transplanting survival rate (81%) reduces on the contrary, and the remarkable processing lower than other; No significant difference between the transplanting survival rate of the different regrowths that obtain of MS concentration and inositol concentration, all reaches 95% left and right.Therefore comprehensive rooting rate, radical, root are grown, root is thick and transplanting survival rate, suitable root media is 1/4MS+NAA 0.1 mg/L, if consideration cost factor, can not add inositol, 1/4MS+NAA 0.1 mg/L that does not add inositol is best root media, takes root and can reach 98%, and the number of taking root is 6.1, length and the rugosity of root are moderate, and transplanting survival rate can reach 96%.
Claims (5)
1. the tissue of Agrimony is cultivated and method for quickly breeding, it is characterized in that the method comprises the following steps:
1) sterilization of explant:
Get the larger leaflet in firm fully-developed Agrimony winglike compound leaf, add washing agent and carefully scrub, then rinse with flowing water, in super-clean bench, blade is proceeded in alcoholic solution and soaked, after proceed to soaking disinfection in thimerosal, with aseptic water washing, then blade is cut into 1 ~ 3 cm
2fritter, be inoculated into and add on the MS medium of plant growth regulating substance with heads horizontal disposing way;
2) adventitious bud inducing
The plant growth regulating substance adding in the MS medium of explant access composed as follows:
TDZ 0.3 mg/L +6-BA 2.0 mg/L；
First secretly cultivate, temperature is 25 ± 1 DEG C, and condition of culture is light/dark hour than being 12 ~ 16:8 ~ 12, intensity of illumination 30 ~ 40 μ mol/(m
2s);
3) adventitious bud proliferation
By the indefinite bud clump inducing in medium, be cut into the fritter of 2 ~ 3 strains, inoculation is additionally added in the medium of IAA0.2 ~ 2.0 mg/L again;
4) cut that blade is light green, the indefinite bud vigorous and that 2 ~ 3 cm are high of growing, be inserted in root media, described root media comprises:
Minimal medium 1/4 MS ~ MS
NAA 0.05~0.5 mg/L；
When hardening, crack cultivation bottle cap carries out hardening, and the medium that after 20 ~ 30h, taking-up clean root adhere to moves in soil, and the initial stage will be carried out moist keeping measures, and In Shade, treats that young leaves grows, and can remove beaker.
2. the tissue of Agrimony according to claim 1 is cultivated and method for quickly breeding, it is characterized in that step 1) thimerosal is the 0.2%HgCl that drips a 1 mol/L NaOH
2solution.
3. the tissue of Agrimony according to claim 2 is cultivated and method for quickly breeding, it is characterized in that step 1) disinfecting time is 5 ~ 6 min.
4. the tissue of Agrimony according to claim 1 is cultivated and method for quickly breeding, it is characterized in that in step 4) root media, NAA is 0.08 ~ 0.3mg/L.
5. the tissue of Agrimony according to claim 1 is cultivated and method for quickly breeding, it is characterized in that step 4) root media is 1/4MS+NAA 0.1 mg/L.
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| CN104813940B (en) * | 2015-05-18 | 2017-05-31 | 中国计量学院 | A kind of quick breeding method for tissue culture of Chinese asystasiella herb |
| CN105961202B (en) * | 2016-05-26 | 2018-03-16 | 上海市园林科学规划研究院 | A kind of rapid propagation method of hairyvein agrimony |
| CN107853129A (en) * | 2017-11-24 | 2018-03-30 | 浙江省农业科学院 | A kind of strawberry cultivation substrate |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000057690A2 (en) * | 1999-03-25 | 2000-10-05 | University Of Guelph | Micropropagation and production of phytopharmaceutical plants |
| CN101268758A (en) * | 2008-05-09 | 2008-09-24 | 南京林业大学 | Quick replication method for Xuan pawpaw tissue cultivation |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000057690A2 (en) * | 1999-03-25 | 2000-10-05 | University Of Guelph | Micropropagation and production of phytopharmaceutical plants |
| CN101268758A (en) * | 2008-05-09 | 2008-09-24 | 南京林业大学 | Quick replication method for Xuan pawpaw tissue cultivation |
Non-Patent Citations (2)
| Title |
|---|
| 吴美芬.龙芽草的组织培养.《植物生理学通讯》.1986,(第4期),第56页. |
| 龙芽草的组织培养;吴美芬;《植物生理学通讯》;19860415(第4期);第56页左栏第1段至右栏第4段 * |
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