CN105210864A - A kind of acquisition methods of dahlia detoxic seedling - Google Patents
A kind of acquisition methods of dahlia detoxic seedling Download PDFInfo
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- CN105210864A CN105210864A CN201510569329.4A CN201510569329A CN105210864A CN 105210864 A CN105210864 A CN 105210864A CN 201510569329 A CN201510569329 A CN 201510569329A CN 105210864 A CN105210864 A CN 105210864A
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Abstract
The invention discloses a kind of acquisition methods of dahlia detoxic seedling, it is characterized in that: the method step is as follows: 1) gather dahlia tender leaf, dedifferentiation produces callus; 2) callus is heat-treated, then callus is continued to induce indefinite bud, adventitious bud rooting is trained healthy and strong plantlet in vitro; 3) peel off plantlet in vitro stem apex further, dedifferentiation produces callus; 4) again callus is heat-treated rear induction seedling; 5) randomly draw the plantlet in vitro in step 4), carry out Viral diagnosis, if detect qualified i.e. dahlia virus-elimination seedlings, need 1 be repeated if defective) to 4) step, until obtain qualified dahlia virus-elimination seedlings.The present invention produces callus breaking up after callus thermal treatment again by twice dedifferentiation, finally obtains dahlia detoxification seedling, solve the detoxification of existing detoxification technology not thoroughly, the difficult problem of complex operation, the needs that batch production detoxification is produced can be met.
Description
Technical field
The present invention relates to a kind of acquisition methods of dahlia detoxic seedling, be specifically related to a kind of produce callus by twice dedifferentiation and will break up again after callus thermal treatment, the final efficient method obtaining dahlia detoxification seedling.
Background technology
Dahlia (
dahliapinnatacav) be the perennial flowering bulb of composite family dahlia, full genus about 27 kinds, have another name called Dali chrysanthemum, dahlia, passionflower, foreign Chinese herbaceous peony, large beautiful chrysanthemum, pachyrhizus, sweet potato flower etc., originate in the state such as Mexico, Colombia and Guatemala, after pass to Europe, Japan and China.Dahlia cane is sturdy, and blade is loose, and flower shape is similar to tree peony, spends large and beautiful in colour, has the features such as colored appearance is graceful, flower pattern is various, the florescence is long, various in style, is described as the favorite in flowers.Dahlia is natural interspecific cross, and the history of artificially breeding is not long, and countries in the world are generally cultivated, and at present northeastward, northwest, the cultivation of the ground such as North China be comparatively extensive, wherein many at Guangdong autumn, winter two season cultivation.Select through artificial hybridization, become allooctaploid, new varieties emerge in an endless stream, and pattern is delicate and charming, and the florescence is long, strong adaptability, are easy to cultivation, therefore suddenly become world's famous flower.Current dahlia is the city flower of Mexican national flower, Seattle, and the province in Jilin Province of China spends, the city flower in the city such as Zhangjiakou, Chifeng, packet header (little beautiful flower), and dahlia symbol is generous, gorgeous, and good luck is world-renowned ornamental flower.
Dahlia adopts point root and cottage propagation usually, but point root and cottage propagation reproduction coefficient low, but every strain is often only and can be separated 5-6 new strain, the requirement that industrialization is produced cannot be met, and use division propagation always, easily make virus by generation accumulation, thus cause dahlia deterioration of variety serious.
Utilize the technology such as tissue cultures, rapid propagation in vitro to carry out stem apex detoxify cultivation, the problem of dahlia virus disease can be solved.More domestic researchers adopt dahlia explant, carry out the research of tissue cultures and detoxification technology, obtain some progress, but the tissue cultures of dahlia and detoxification technology studies in China are started late, research accumulation is few, the detoxification technology of current dahlia need progressive, detoxification efficiency is low, complex operation has seriously hindered the raising of dahlia quality and the demand of production, therefore tissue cultures is carried out to dahlia, rapid propagation in vitro technical research, set up and optimize a kind of detoxic seedling Cultivating techniques efficiently, there are great industrial value and the wide prospect of marketing.
Summary of the invention
The object of the invention is the deficiency existed for existing dahlia detoxification technology; a kind of sustainability, easy to operate and can the method for efficient acquisition dahlia detoxic seedling of large-scale production is provided; the detoxification this method solving existing detoxification technology not thoroughly, the difficult problem of complex operation, the needs that batch production detoxification is produced can be met.
The object of the invention is to solve by the following technical programs:
An acquisition methods for dahlia detoxic seedling, is characterized in that: the method step is as follows:
1) gather dahlia tender leaf, dedifferentiation produces callus;
2) callus is heat-treated, then callus is continued to induce indefinite bud, adventitious bud rooting is trained healthy and strong plantlet in vitro;
3) peel off plantlet in vitro stem apex further, dedifferentiation produces callus;
4) again callus is heat-treated rear induction seedling;
5) randomly draw the plantlet in vitro in step 4), carry out Viral diagnosis, if detect qualified i.e. dahlia virus-elimination seedlings, need 1 be repeated if defective) to 4) step, until obtain qualified dahlia virus-elimination seedlings.
The detailed step of the method is as follows:
1) gather dahlia tender leaf, dedifferentiation produces callus:
Gather the tender leaf growing to the anosis dahlia of the health in squaring period, running water is cleaned, running water 30min, with 70% alcohol-pickled 15s, in 0.1% mercuric chloride, addend drips Tween-20 solution and soaks 15min, and stir with glass bar, with aseptic water washing 4-5 time, filter paper blots, and punches with the card punch of bore dia 5mm, by the blade inoculation of laying to light culture under DEG C condition of 25-27 on calli induction media, within 10-15 days, produce the callus that pistac is loose afterwards;
2) callus is heat-treated, then callus is continued to induce indefinite bud, adventitious bud rooting is trained healthy and strong plantlet in vitro:
To large grain of rice size be grown to, transfer to thermal treatment in incubator with the callus of a small amount of explant, after excising brownization block section, callus is transferred to light cultivation under 25-27 DEG C of condition on adventitious bud induction culture base, after 20-30 days, Calli Differentiation goes out indefinite bud, the bud of more than height 1cm born is again forwarded to root media and is trained healthy and strong plantlet in vitro;
3) peel off plantlet in vitro stem apex further, dedifferentiation produces callus:
Treat that plantlet in vitro grows to 5cm-8cm, the shoot tip meristem stripping 0.4-0.6cm size is inoculated on Stem tip induction callus medium, and under 25-27 DEG C of condition, light culture 30-45 days, induces callus;
4) again callus is heat-treated rear induction seedling:
Again stem apex callus is heat-treated, callus is transferred on adventitious bud induction culture base after excising brownization block section, under 25-27 DEG C of condition, light is cultivated after 20-25 days and is induced indefinite bud, then transfers into root media, can obtain dahlia seedling after 40-50 days;
5) randomly draw the plantlet in vitro in step 4), carry out Viral diagnosis, if detect qualified i.e. dahlia virus-elimination seedlings, need 1 be repeated if defective) to 4) step, until obtain qualified dahlia virus-elimination seedlings:
Randomly draw 8-10 only complete step 4) dahlia blade, carry out the detection of RT-PCR retroviruse, if detect qualified i.e. dahlia virus-elimination seedlings, need 1 be repeated if defective) to 4) step, until obtain qualified dahlia virus-elimination seedlings.
Calli induction media formula for inducing dahlia blade to produce callus in step 1) is: MS+1.5mg/L6-BA+0.3mg/LNAA+3% sucrose+6.5g/L agar, pH is 5.8.
Step 2) in adventitious bud induction culture based formulas that just callus continues to induce indefinite bud be: MS+2.5mg/L6-BA+0.08NAA+3% sucrose+6.5g/L plant gel, pH is 5.8; Described illumination condition callus being continued induce indefinite bud, adventitious bud rooting is cultivated is: intensity of illumination is 1500-2000Lux, and the photoperiod is that 14h light/10h is dark.
Step 2) and step 4) in heat treatment method be: to hocket 2-3 circulation at 38 DEG C of-42 DEG C of high temperature treatment 6h-8h, then 26 DEG C of-28 DEG C of low temperature treatment 10h-12h.
Culture medium prescription for inducing stem apex to produce callus in step 3) is: MS+4.5mg/L6-BA+0.5mg/LNAA+0.5mg/LKT+3% sucrose+6.5g/L agar, pH is 5.8.
The adventitious bud induction culture based formulas in step 4), callus induction being gone out indefinite bud is: MS+1.5mg/L6-BA+0.3mg/LNAA+3% sucrose+6.5g/L agar, and pH is 5.8; Prescription of rooting medium is: 1/2MS+0.15mg/LNAA+1.5% sucrose+6.5g/L agar, pH is 5.8.
The illumination condition being continued by stem apex callus in step 4) to induce indefinite bud, adventitious bud rooting is cultivated is: intensity of illumination is 1500-2000Lux, and the photoperiod is that 12h light/12h is dark.
The viral species of step 5) Viral diagnosis is dahlia mosaic virus, tobacco mosaic virus and cucumber mosaic virus.
The acquisition methods of a kind of dahlia detoxic seedling of the present invention, be the preferred result that inventor obtains through carefully studying, after lot of experiment validation, compared to existing technology, innovative point of the present invention is:
The present invention is by adopting comprehensive detoxification technology, the i.e. method that combines of twice callus induction, callus thermal treatment detoxification, stem apex detoxify, effectively overcome the difficult problems such as detoxification material is more, detoxification operating process is loaded down with trivial details, detoxification inefficiency, efficiently can obtain dahlia detoxification seedling.
The invention have employed the heat treated poison-removing method of callus, after adopting callus thermal treatment of the present invention, cut away brownization part, the unconspicuous callus part of brownization degree after cultivation thermal treatment, effectively can get rid of the virus that callus carries, substantially increase detoxification efficiency, and avoid sterile working, simple to operate.
The acquisition methods of dahlia detoxic seedling of the present invention adopts Stem tip induction to become callus; improve the kind of dahlia, be stripped of dahlia virus further again, the method flow process simply, easily operates; therefore be easy to the virus-elimination seedlings forming large-scale production, be suitable for promoting the use of.
Accompanying drawing explanation
Fig. 1 is the result figure that dahlia dedifferentiation produces callus;
Fig. 2 is the result figure that dahlia Calli Differentiation goes out indefinite bud;
Fig. 3 is the dahlia seedling result figure after stem apex detoxify after culture of rootage;
Fig. 4 is the comparing result figure of juvenile traits before and after dahlia detoxification.
Embodiment
Below in conjunction with specific embodiments and the drawings, the present invention is further illustrated.
An acquisition methods for dahlia detoxic seedling, the detailed step of the method is as follows:
1) tender leaf of dahlia is gathered, dedifferentiation produces callus: gather the tender leaf growing to the anosis dahlia of the health in squaring period, running water is cleaned, running water 30min, with 70% alcohol-pickled 15s, in 0.1% mercuric chloride, addend drips Tween-20 solution and soaks 15min, and stir with glass bar, with aseptic water washing 5 times, filter paper blots, punch with the card punch of bore dia 4mm, by the blade inoculation of laying, to calli induction media, (culture medium prescription is: MS+1.5mg/L6-BA+0.3mg/LNAA+3% sucrose+6.5g/L agar, pH is 5.8) light culture under upper 25-27 DEG C of condition, after 13 days, we observe the appearance of the callus that pistac is loosened, as shown in Figure 1,
2) callus is heat-treated, then callus is continued to induce indefinite bud, adventitious bud rooting is trained healthy and strong plantlet in vitro: will grow to large grain of rice size, callus with a small amount of explant is transferred in constant incubator, 40 DEG C of heat treatment process 7h, then 27 DEG C of low temperature treatment 11h, hocket and circulate for 3 times, then (culture medium prescription is: MS+2.5mg/L6-BA+0.08NAA+3% sucrose+6.5g/L plant gel after excising brownization block section, callus to be transferred to adventitious bud induction culture base, pH is 5.8) light is cultivated under upper 25-27 DEG C of condition, the intensity of illumination that light is cultivated controls as 1800Lux, photoperiod controls as 14h light/10h is dark, Calli Differentiation can be observed after 25 days and go out indefinite bud, as shown in Figure 2, the bud of more than height 1cm born is again forwarded to root media and is trained healthy and strong plantlet in vitro,
3) plantlet in vitro stem apex is peeled off further, dedifferentiation produces callus: treat that plantlet in vitro grows to 5cm-8cm, the shoot tip meristem stripping 0.4-0.6cm size is inoculated on Stem tip induction callus medium that (culture medium prescription is: MS+4.5mg/L6-BA+0.5mg/LNAA+0.5mg/LKT+3% sucrose+6.5g/L agar, pH is 5.8), then by stem apex light culture about 32 days under 25-27 DEG C of condition under 25-27 DEG C of condition, callus is induced;
4) again callus is heat-treated rear induction seedling: again heat-treated by stem apex callus, heat treatment method is with step 2), (culture medium prescription is: MS+1.5mg/L6-BA+0.3mg/LNAA+3% sucrose+6.5g/L agar after excising brownization block section, callus to be transferred to adventitious bud induction culture base, pH is 5.8) on, intensity of illumination is 1500-2000Lux, photoperiod is the illumination condition that 12h light/12h is dark, cultivate under 25-27 DEG C of temperature condition, within about 21 days, adventitious bud inducing evenly occurs, then transferring into root media, (culture medium prescription is: 1/2MS+0.15mg/LNAA+1.5% sucrose+6.5g/L agar, pH is 5.8), within about 40 days, induce dahlia seedling, seedling active growth after 50 days, as Fig. 3,
5) plantlet in vitro in step 4) is randomly drawed, carry out Viral diagnosis: randomly draw the dahlia blade that 10 complete step 4), carry out the detection of RT-PCR retroviruse, the viral species of detection is dahlia mosaic virus, tobacco mosaic virus and cucumber mosaic virus, do not find that virus is residual, show the thoroughly detoxification of dahlia seedling, this seedling is expanded numerous in vitro, obtain a large amount of detoxification seedling, detoxic seedling blade is unfolded, Miao Zhuan, the more non-detoxic seedling of growing way improves, greatly as Fig. 4.
The present invention is by adopting comprehensive detoxification technology, the i.e. method that combines of twice callus induction, callus thermal treatment detoxification, stem apex detoxify, effectively overcome the difficult problems such as detoxification material is more, detoxification operating process is loaded down with trivial details, detoxification inefficiency, efficiently can obtain dahlia detoxification seedling.The invention have employed the heat treated poison-removing method of callus, after adopting callus thermal treatment of the present invention, cut away brownization part, the unconspicuous callus part of brownization degree after cultivation thermal treatment, effectively can remove the virus that callus carries, substantially increase detoxification efficiency, and decrease sterile working, simple to operate.The method of efficient acquisition dahlia detoxification seedling of the present invention adopts Stem tip induction to become callus; improve the kind of dahlia, be stripped of dahlia virus further again, the method flow process simply, easily operates; therefore be easy to the virus-elimination seedlings forming large-scale production, be suitable for promoting the use of.
Claims (9)
1. an acquisition methods for dahlia detoxic seedling, is characterized in that: the method step is as follows:
1) gather dahlia tender leaf, dedifferentiation produces callus;
2) callus is heat-treated, then callus is continued to induce indefinite bud, adventitious bud rooting is trained healthy and strong plantlet in vitro;
3) peel off plantlet in vitro stem apex further, dedifferentiation produces callus;
4) again callus is heat-treated rear induction seedling;
5) randomly draw the plantlet in vitro in step 4), carry out Viral diagnosis, if detect qualified i.e. dahlia virus-elimination seedlings, need 1 be repeated if defective) to 4) step, until obtain qualified dahlia virus-elimination seedlings.
2. the acquisition methods of dahlia detoxic seedling according to claim 1, is characterized in that: the detailed step of the method is as follows:
1) gather dahlia tender leaf, dedifferentiation produces callus:
Gather the tender leaf growing to the anosis dahlia of the health in squaring period, running water is cleaned, running water 30min, with 70% alcohol-pickled 15s, in 0.1% mercuric chloride, addend drips Tween-20 solution and soaks 15min, and stir with glass bar, with aseptic water washing 4-5 time, filter paper blots, and punches with the card punch of bore dia 5mm, by the blade inoculation of laying to light culture under DEG C condition of 25-27 on calli induction media, within 10-15 days, produce the callus that pistac is loose afterwards;
2) callus is heat-treated, then callus is continued to induce indefinite bud, adventitious bud rooting is trained healthy and strong plantlet in vitro:
To large grain of rice size be grown to, transfer to thermal treatment in incubator with the callus of a small amount of explant, after excising brownization block section, callus is transferred to light cultivation under 25-27 DEG C of condition on adventitious bud induction culture base, after 20-30 days, Calli Differentiation goes out indefinite bud, the bud of more than height 1cm born is again forwarded to root media and is trained healthy and strong plantlet in vitro;
3) peel off plantlet in vitro stem apex further, dedifferentiation produces callus:
Treat that plantlet in vitro grows to 5cm-8cm, the shoot tip meristem stripping 0.4-0.6cm size is inoculated on Stem tip induction callus medium, and under 25-27 DEG C of condition, light culture 30-45 days, induces callus;
4) again callus is heat-treated rear induction seedling:
Again stem apex callus is heat-treated, callus is transferred on adventitious bud induction culture base after excising brownization block section, under 25-27 DEG C of condition, light is cultivated after 20-25 days and is induced indefinite bud, then transfers into root media, can obtain dahlia seedling after 40-50 days;
5) randomly draw the plantlet in vitro in step 4), carry out Viral diagnosis, if detect qualified i.e. dahlia virus-elimination seedlings, need 1 be repeated if defective) to 4) step, until obtain qualified dahlia virus-elimination seedlings:
Randomly draw 8-10 only complete step 4) dahlia blade, carry out the detection of RT-PCR retroviruse, if detect qualified i.e. dahlia virus-elimination seedlings, need 1 be repeated if defective) to 4) step, until obtain qualified dahlia virus-elimination seedlings.
3. the acquisition methods of dahlia detoxic seedling according to claim 1 and 2, it is characterized in that: the calli induction media formula for inducing dahlia blade to produce callus in step 1) is: MS+1.5mg/L6-BA+0.3mg/LNAA+3% sucrose+6.5g/L agar, pH is 5.8.
4. the acquisition methods of dahlia detoxic seedling according to claim 1 and 2, it is characterized in that: step 2) in adventitious bud induction culture based formulas that just callus continues to induce indefinite bud be: MS+2.5mg/L6-BA+0.08NAA+3% sucrose+6.5g/L plant gel, pH is 5.8; Described illumination condition callus being continued induce indefinite bud, adventitious bud rooting is cultivated is: intensity of illumination is 1500-2000Lux, and the photoperiod is that 14h light/10h is dark.
5. the acquisition methods of dahlia detoxic seedling according to claim 1 and 2, is characterized in that: step 2) and step 4) in heat treatment method be: to hocket 2-3 circulation at 38 DEG C of-42 DEG C of high temperature treatment 6h-8h, then 26 DEG C of-28 DEG C of low temperature treatment 10h-12h.
6. the acquisition methods of dahlia detoxic seedling according to claim 1 and 2, it is characterized in that: the culture medium prescription for inducing stem apex to produce callus in step 3) is: MS+4.5mg/L6-BA+0.5mg/LNAA+0.5mg/LKT+3% sucrose+6.5g/L agar, pH is 5.8.
7. the acquisition methods of dahlia detoxic seedling according to claim 1 and 2, it is characterized in that: the adventitious bud induction culture based formulas in step 4), callus induction being gone out indefinite bud is: MS+1.5mg/L6-BA+0.3mg/LNAA+3% sucrose+6.5g/L agar, pH is 5.8; Prescription of rooting medium is: 1/2MS+0.15mg/LNAA+1.5% sucrose+6.5g/L agar, pH is 5.8.
8. the acquisition methods of dahlia detoxic seedling according to claim 1 and 2, it is characterized in that: the illumination condition being continued by stem apex callus in step 4) to induce indefinite bud, adventitious bud rooting is cultivated is: intensity of illumination is 1500-2000Lux, and the photoperiod is that 12h light/12h is dark.
9. the acquisition methods of dahlia detoxic seedling according to claim 1 and 2, is characterized in that: the viral species of step 5) Viral diagnosis is dahlia mosaic virus, tobacco mosaic virus and cucumber mosaic virus.
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Cited By (3)
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| CN106818487A (en) * | 2017-02-24 | 2017-06-13 | 宋立胜 | A kind of screening technique of paddy rice high temperature resistant mutant strain |
| CN111642392A (en) * | 2020-04-29 | 2020-09-11 | 上海润绣农业科技有限公司 | Sterilization method for surface of dahlia explant |
| CN115644009A (en) * | 2022-11-02 | 2023-01-31 | 云南省农业科学院花卉研究所 | Method for annual production of fresh cut flowers by regulating and controlling flowering period of dahlia |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106818487A (en) * | 2017-02-24 | 2017-06-13 | 宋立胜 | A kind of screening technique of paddy rice high temperature resistant mutant strain |
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| CN111642392A (en) * | 2020-04-29 | 2020-09-11 | 上海润绣农业科技有限公司 | Sterilization method for surface of dahlia explant |
| CN115644009A (en) * | 2022-11-02 | 2023-01-31 | 云南省农业科学院花卉研究所 | Method for annual production of fresh cut flowers by regulating and controlling flowering period of dahlia |
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Application publication date: 20160106 |