CN109287489B - Standardized in-vitro culture method of pyrethrum seedlings - Google Patents
Standardized in-vitro culture method of pyrethrum seedlings Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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Abstract
The invention provides a method for standardized isolated culture of pyrethrum seedlings, which is mainly characterized in that buds and seeds which are not opened of pyrethrum are used as explant materials for isolated culture, the buds and the seeds are sterilized and disinfected, the buds are inoculated on a special induction culture medium for the buds, the seeds are inoculated on the special induction culture medium for the seeds for induction, and then proliferation, propagation, rooting culture and hardening and transplanting are carried out. The method can be standardized and accurately operated in each step, and standardized production of pyrethrum is established and realized, so that the method is suitable for industrial production. The seedlings obtained by the method can maintain the excellent characters of the pyrethrum female parent, are not limited by the time of taking the explants, and have high propagation speed and high survival rate.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for standardized in-vitro culture of pyrethrum seedlings.
Background
Pyrethrum (Tanacetum cinerriifolium) is a perennial herb of the family Compositae, also known as pyrethrum flower. Pyrethrum is a good raw material plant of biological pesticide, the planting area in Yunnan is about 10 ten thousand mu, account for the 30% of the world's output. Pyrethrin can be extracted from flowers of the plants, and has a plurality of advantages, such as capability of quickly paralyzing and knocking down pests such as mosquitoes, flies, fleas, lice and the like; the material is easy to be naturally decomposed under high temperature and strong light; has no toxic and side effects on human beings, livestock and the like; no pollution to the environment; is not easy to generate resistance to the use of pests. In addition, the pyrethrum flowers can be used as a medicine, tinea can be squeezed out by external application of skin, the pyrethrum flowers are rich and beautiful, the development potential of the ornamental value is huge, and the pyrethrum flowers can be used for arranging flower beds and can also be used as fresh cut flowers.
The currently planted pyrethrum varieties are reserved for years, so that the degeneration is serious, the mixing rate is increased, the quality is seriously reduced, the first production can be held from the source by utilizing the standardized in-vitro preservation and rejuvenation technology, and the standardized production of the pyrethrum is realized.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for standardized isolated culture of pyrethrum seedlings, which comprises the following steps:
(1) taking explant materials and sterilizing: selecting buds and seeds of the excellent pyrethrum varieties which are not opened as explant materials, wherein the selected explant materials are required to be robust, free of diseases and insect pests; the selected explant material is sterilized and disinfected in sequence according to the following method: washing the explant material with 3% w/w laundry powder water, placing the washed explant material into running water for running water to wash for 10-20min, disinfecting the explant material with 70% v/v alcohol for 30s, running water for running water to wash for 10-20min, placing the explant material into 0.1% w/w mercuric chloride solution for soaking and disinfecting for 10-15 min, continuously stirring and oscillating in the soaking and disinfecting process, taking the explant material out of the mercuric chloride solution, soaking the explant material with 15% w/w sodium hypochlorite solution for 10-20min, continuously stirring and oscillating in the soaking process, taking the explant material out of the sodium hypochlorite solution after soaking, washing the explant material with sterile water for 3-5 times, and inoculating the washed explant material for later use;
(2) inducing cluster buds: placing the washed explant material into an ultraclean workbench, cutting the browned part of the explant by using an inoculating knife, and inducing cluster buds of the bud explant and the seed explant in the following way:
inoculating the bud explant to a special induction culture medium for buds, and culturing for 20-30 days to obtain pyrethrum cluster seedlings, wherein the special induction culture medium for buds is as follows: MS +6-BA2.0mg/L + TDZ 0.5mg/L + IBA 0.2mg/L + agar 7g/L + sucrose 30g/L, pH value is 5.6, and culture conditions are as follows: the temperature is 25 +/-1 ℃, the illumination intensity is 1000-;
inoculation of seed explants to seed specific InductionCulturing in dark for 7 days, and culturing under illumination for 15-30 days to obtain cluster seedlings of pyrethrum: the temperature is 25 +/-1 ℃, the illumination intensity is 1000-: MS + GA 1.0mg/L + TDZ 0.1mg/L + IAA0.1mg/L + AgNO31.0mg/L + agar 7g/L + sucrose 30g/L, pH 5.6;
(3) and (3) sterile seedling proliferation: transferring the cluster pyrethrum seedlings obtained by inducing the bud explants and the seed explants in the step (2) to an aseptic seedling proliferation culture medium under the conditions of temperature and light: culturing at 25 +/-1 ℃ under the illumination intensity of 1000-2000lx for 12h/d for 20-25 days to obtain differentiated and expanded pyrethrum clump seedlings, and repeating the sterile seedling proliferation process to expand and propagate to obtain a target number of pyrethrum clump seedlings; the sterile seedling multiplication medium comprises: MS +6-BA 1.0mg/L + TDZ 0.1mg/L + IBA0.1mg/L + agar 7g/L + sucrose 30g/L, pH 5.6;
(4) rooting culture: cutting the cluster seedlings of the pyrethrum obtained by propagation and propagation in the step (4) into single seedlings by using an inoculating knife, transferring the cut single seedlings to a pyrethrum rooting culture medium, and performing the following steps under the conditions of temperature and light: culturing at 25 +/-1 ℃ under the illumination intensity of 1000-2000lx for 12h/d for 15-20 days to obtain pyrethrum rooting seedlings; the pyrethrum rooting culture medium comprises: MS + IAA0.1mg/L + IBA0.1mg/L + agar 7g/L + sucrose 30g/L, pH 5.6;
(5) hardening seedlings: taking out the pyrethrum rooted seedlings from the culture bottle, washing off the culture medium at the roots of the pyrethrum rooted seedlings, and transplanting the pyrethrum rooted seedlings, from which the culture medium is washed off, to turf: hardening seedlings in the mixed matrix with the volume ratio of 6:1 of perlite, and transplanting the rooting seedlings of the pyrethrum to a field after the rooting seedlings of the pyrethrum grow to 20-30 cm.
Compared with the prior art, the invention has the following beneficial effects:
standardizing the in vitro culture method of pyrethrum seedlings, performing standardized and accurate operation from the steps of induction, proliferation, rooting and the like, establishing and realizing standardized production of pyrethrum, and being suitable for industrialized production. The seedlings obtained by the method can keep the excellent characters of the female parent of the pyrethrum, and provide excellent seed sources for the planting of the pyrethrum, thereby enhancing the core competitiveness of the pyrethrum in the international market in China, improving the overall quality level of products, and promoting the quality improvement and efficiency improvement of the industry and the earning of exports.
The method expands the explant material of the pyrethrum in vitro propagation, can provide seedlings for customers for cultivating the pyrethrum at any time, is not limited by the time for obtaining the explant, has high propagation speed, the differentiation and propagation speed of each time can reach 4-5 times, has high survival rate, solves the problem of low seedling raising efficiency of pyrethrum seeds, and has important practical significance and application prospect for promoting the production of the pyrethrum seedlings.
Detailed Description
The present invention will be described in detail with reference to examples, which are not specifically described but are conventional methods.
Examples
(1) Taking explant materials and sterilizing: selecting buds and seeds of the excellent pyrethrum varieties which are not opened as explant materials, wherein the selected explant materials are required to be robust, free of diseases and insect pests; the selected explant material is sterilized and disinfected in sequence according to the following method: washing the explant material with 3% w/w laundry powder water, placing the washed explant material into tap water, flushing the washed explant material with running water for 20min, disinfecting the explant material with 70% v/v alcohol for 30s, flushing the explant material with running water for 10-20min, placing the explant material in 0.1% w/w mercuric chloride solution, soaking and disinfecting for 10min, continuously stirring and oscillating in the soaking and disinfecting process, taking the explant material out of the mercuric chloride solution, soaking the explant material with 15% w/w sodium hypochlorite solution for 10min, continuously stirring and oscillating in the soaking process, taking the explant material out of the sodium hypochlorite solution after soaking, flushing the explant material with sterile water for 3-5 times, and inoculating the flushed explant material for later use;
(2) inducing cluster buds: placing the washed explant material into a clean bench, cutting the slightly browned part of the explant by using an inoculating knife, and inducing cluster buds of the bud explant and the seed explant in the following way:
inoculating the bud explant to a special induction culture medium for buds, and culturing for 20 days to obtain pyrethrum cluster seedlings, wherein the special induction culture medium for buds is as follows: MS +6-BA2.0mg/L + TDZ 0.5mg/L + IBA 0.2mg/L + agar 7g/L + sucrose 30g/L, pH value is 5.6, and culture conditions are as follows: the temperature is 25 +/-1 ℃, the illumination intensity is 1000-;
inoculating the seed explant to a special induction culture medium for seeds, performing dark culture for 7 days, and then transferring to a light culture condition for culture for 15 days to obtain pyrethrum cluster seedlings, wherein the light culture condition is as follows: the temperature is 25 +/-1 ℃, the illumination intensity is 1000-: MS + GA 1.0mg/L + TDZ 0.1mg/L + IAA0.1mg/L + AgNO31.0mg/L + agar 7g/L + sucrose 30g/L, pH 5.6;
(3) and (3) sterile seedling proliferation: transferring the cluster pyrethrum seedlings obtained by inducing the bud explants and the seed explants in the step (2) to an aseptic seedling proliferation culture medium under the conditions of temperature and light: culturing at 25 +/-1 ℃ and with the illumination intensity of 1000-2000lx for 12h/d for 20 days to obtain differentiated and expanded pyrethrum clump seedlings, repeating the sterile seedling proliferation process, and expanding to obtain a target number (large batch) of pyrethrum clump seedlings, wherein the differentiation and expansion speed is 4-5 times each time; the sterile seedling multiplication medium comprises: MS +6-BA 1.0mg/L + TDZ 0.1mg/L + IBA0.1mg/L + agar 7g/L + sucrose 30g/L, pH 5.6;
(4) rooting culture: cutting the cluster seedlings of the pyrethrum obtained by propagation and propagation in the step (4) into single seedlings by using an inoculating knife, transferring the cut single seedlings to a pyrethrum rooting culture medium, and performing the following steps under the conditions of temperature and light: culturing at 25 +/-1 ℃ under the illumination intensity of 1000-2000lx for 12h/d for 15 days to obtain pyrethrum rooting seedlings; the rooting quantity of each seedling is 10-15, the length of each root is 2.0-3.0 cm, and the plant is robust; the pyrethrum rooting culture medium comprises: MS + IAA0.1mg/L + IBA0.1mg/L + agar 7g/L + sucrose 30g/L, pH 5.6.
(5) Hardening seedlings: taking out the pyrethrum rooted seedlings from the culture bottle, washing off the culture medium at the roots of the pyrethrum rooted seedlings, and transplanting the pyrethrum rooted seedlings, from which the culture medium is washed off, to turf (the particle size is 1-5 mm): and (3) hardening the seedlings in a mixed matrix with the volume ratio of 6:1 of perlite (the particle size is 1-5 mm), and transplanting the rooted seedlings of the pyrethrum to a field after the rooted seedlings of the pyrethrum survive and grow to 20-30 cm.
The breeding and propagation speed is high, the differentiation and propagation speed of each time can reach 4-5 times, buds which are not opened are used as explants, seedlings can be induced to emerge after 20 days, after the buds are transferred to a propagation culture medium, propagation and propagation can be carried out every 15-20 days, and the survival rate is as high as more than 90%. Seeds are used as explants, seedlings can be induced after 22 days, after the seedlings are transferred to a propagation culture medium, propagation can be carried out every 15 to 20 days, and the survival rate is up to more than 90 percent.
Claims (1)
1. A method for standardized in-vitro culture of pyrethrum seedlings is characterized by comprising the following steps:
(1) taking explant materials and sterilizing: selecting a bud of a superior pyrethrum variety which is not opened as an explant material, wherein the selected explant material is required to be robust, free of diseases and insect pests; the selected explant material is sterilized and disinfected in sequence according to the following method: washing the explant material with 3% w/w laundry powder water, placing the washed explant material into running water for running water to wash for 10-20min, disinfecting the explant material with 70% v/v alcohol for 30s, running water for running water to wash for 10-20min, placing the explant material into 0.1% w/w mercuric chloride solution for soaking and disinfecting for 10-15 min, continuously stirring and oscillating in the soaking and disinfecting process, taking the explant material out of the mercuric chloride solution, soaking the explant material with 15% w/w sodium hypochlorite solution for 10-20min, continuously stirring and oscillating in the soaking process, taking the explant material out of the sodium hypochlorite solution after soaking, washing the explant material with sterile water for 3-5 times, and inoculating the washed explant material for later use;
(2) inducing cluster buds: placing the washed explant material into an ultraclean workbench, cutting the browned part of the explant by using an inoculating knife, and inducing the cluster buds of the bud explant in the following way: inoculating the bud explant to a special induction culture medium for buds, and culturing for 20-30 days to obtain pyrethrum cluster seedlings, wherein the special induction culture medium for buds is as follows: MS +6-BA2.0mg/L + TDZ 0.5mg/L + IBA 0.2mg/L + agar 7g/L + sucrose 30g/L, the pH value is 5.6, and the culture conditions are as follows: the temperature is 25 +/-1 ℃, the illumination intensity is 1000-;
(3) and (3) sterile seedling proliferation: transferring cluster seedlings of pyrethrum obtained by inducing the bud explants in the step (2) to an aseptic seedling multiplication medium under the conditions of temperature and light: culturing at 25 +/-1 ℃ under the illumination intensity of 1000-2000lx for 12h/d for 20-25 days to obtain differentiated and expanded pyrethrum clump seedlings, and repeating the sterile seedling proliferation process to expand and propagate to obtain a target number of pyrethrum clump seedlings; the sterile seedling multiplication medium comprises: MS +6-BA 1.0mg/L + TDZ 0.1mg/L + IBA0.1mg/L + agar 7g/L + sucrose 30g/L, pH 5.6;
(4) rooting culture: cutting the cluster seedlings of the pyrethrum obtained by propagation and propagation in the step (3) into single seedlings by using an inoculating knife, transferring the cut single seedlings to a pyrethrum rooting culture medium, and under the condition of temperature and light: culturing at 25 +/-1 ℃ under the illumination intensity of 1000-2000lx for 12h/d for 15-20 days to obtain pyrethrum rooting seedlings; the pyrethrum rooting culture medium comprises: MS + IAA0.1mg/L + IBA0.1mg/L + agar 7g/L + sucrose 30g/L, pH 5.6;
(5) hardening seedlings: taking out the pyrethrum rooted seedlings from the culture bottle, washing off the culture medium at the roots of the pyrethrum rooted seedlings, and transplanting the pyrethrum rooted seedlings, from which the culture medium is washed off, to turf: perlite 6:1, hardening the seedlings, and transplanting the rooting seedlings of the pyrethrum to a field after the rooting seedlings of the pyrethrum grow to 20-30 cm.
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| CN1915001A (en) * | 2006-08-31 | 2007-02-21 | 中国药科大学 | Technique for breeding pyrethrum of white flower |
| CN101361456A (en) * | 2008-07-25 | 2009-02-11 | 云南省农业科学院花卉研究所 | Efficient flameray-gerbera propagation production method using excised leaf |
| CN101578959B (en) * | 2009-06-24 | 2014-11-12 | 华中农业大学 | Method for isolated culture of pyrethrum regeneration plant and special culture medium |
| CN101810144B (en) * | 2010-05-05 | 2012-08-01 | 北京林业大学 | Rapid breeding method of senecio cruentus |
| CN102783418B (en) * | 2012-08-21 | 2013-09-04 | 云南南宝生物科技有限责任公司 | Tissue culture method for pyrethrum cinerariifolium |
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| CN1918960A (en) * | 2006-08-25 | 2007-02-28 | 贵州芊芊园艺新技术发展公司 | Standard planting method of medicinal lycoris radiata |
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