CN102217540A - Quick propagation method for lycoris chinensis - Google Patents
Quick propagation method for lycoris chinensis Download PDFInfo
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Abstract
The invention discloses a quick propagation method for lycoris chinensis. The method comprises the following steps of: taking embryos of the lycoris chinensis, and inoculating the embryos to an induction medium to induce callus; transferring the callus to an MS (Murashige and Skoog) culture medium containing sucrose, naphthyl acetic acid (NAA) and 6-benzyl aminopurine (6-BA) to induce adventitious buds; transferring the adventitious buds to the MS culture medium containing the sucrose, the NAA and the 6-BA to induce bulblets; transferring the expanded bulblets to an MS culture medium containing agar and NAA, and performing induced rooting under illumination; and transferring the rooted bulbs to a transplanting medium of perlite and humus. According to the method disclosed by the invention, the embryos of the lycoris chinensis are used as explants, the explants are induced to generate the callus, then the adventitious buds and the bulblets are induced, and plant regeneration is finally completed, so that a new path is increased for quick propagation of tissue culture of the lycoris chinensis. The method has high propagation coefficient, millions of bulblets can be produced in one year, and an effective path can be provided for quickly propagating seed bulbs; and the test tube plantlets have high differentiation synchronizing degree and good consistency, and the method has high large-scale production development value and good economic prospect.
Description
Technical field
The present invention relates to the method for quickly breeding of short-tube lycoris, be specifically related to a kind of method with tissue-culturing quick-propagation China short-tube lycoris.
Background technology
Lycoris plants belongs to bulbous plant, and the whole world has 20 kinds approximately, and China has 15 kinds, accounts for the branch 3/4ths of whole world sum.This platymiscium pattern is gorgeous, and the flower type is peculiar, is quite rising fresh cut-flowers material; Because it is in winter-spring season leafing growth, be the rare Winter-Spring ground cover plant in area, the middle and lower reach of Yangtze River; Simultaneously the lycoris plants napiform root contains the valuable bio-pharmaceuticals raw material galanthamine, lycorine of high level etc., be the good medicine of treatment senile dementia disease, so lycoris plants has high economic worth.
China short-tube lycoris (lycoris chinensis) is a kind of lycoris plants, originates in China, is that a kind of collection is viewed and admired, medicine is used for one, and contains the economic class plant of enriching starch.Because its economic development value height, market demand increases day by day.At present it also is in wild state, only relies on to excavate wild species ball natural resources and not only can not meet the need of market, and ecotope has also been caused very big destruction.This shows, plant the bottleneck that the ball breeding problem has become its industrialized development.Breeding kind of ball fast by tissue culture method is the effective way that solves this bottleneck problem.
The breeding of lycoris plants mainly relies on nature bulb separation, scale breeding etc. except that seminal propagation, reproduction coefficient is lower, is difficult to meet the need of market.The quick breeding of adopting tissue culture method to carry out short-tube lycoris is to solve kind of the effective way of ball breeding.Research great majority about the lycoris plants tissue culture concentrate on safflower short-tube lycoris (lycoris radiata), chrysanthemum short-tube lycoris (lycoris aurea) and Bulbus Lycoridis longitubae (lycoris longituba) and the red blue stone garlic (lycoris haywardii), and the method for tissue culture of employing is to realize plant regeneration with the bulb for the explant induction indefinite bud mostly.It is explant that Zhu Jin (2000), Wang Yan (2007) adopt the scale of safflower short-tube lycoris; Guo Zhaowu (2010), Lichun-Huang(1989) be explant with the bulb of chrysanthemum short-tube lycoris; Wang Guangping (2004) is an explant with the Bulbus Lycoridis longitubae bulb; Zhang Haizhen (2008) is an explant with red blue stone garlic bulb, more than all be to be that explant is realized plant regeneration with the bulb; Zhao Zhimin (2009) is that explant induction has produced callus with chrysanthemum short-tube lycoris bulb, but do not finish bud, the formation of clove and plant regeneration.Up to the present, few to the research of Chinese short-tube lycoris, also be not specially adapted to the method for breeding Chinese short-tube lycoris fast.
Summary of the invention
Goal of the invention: at the deficiencies in the prior art, the method for quickly breeding that the purpose of this invention is to provide a kind of Chinese short-tube lycoris, employing is bred bulb in a large number by the method that produces for the explant induction callus with the embryo, provides new way for breeding kind of a ball fast.
Technical scheme: in order to realize the foregoing invention purpose, the technical solution used in the present invention is as follows:
The method for quickly breeding of a kind of Chinese short-tube lycoris may further comprise the steps:
(1) gets the embryo of Chinese short-tube lycoris, be inoculated in inducing culture, the dark evoked callus down of cultivating; Wherein, inducing culture is the MS that contains the MS of 2,4 dichlorophenoxyacetic acid 2.0 ~ 3.0mg/L or contain 6-benzyl purine 0.1 ~ 0.5mg/L, 2,4 dichlorophenoxyacetic acid 1.0 ~ 2.0mg/L, caseinhydrolysate 250 ~ 500mg/L;
(2) callus of step (1) is transferred in the MS that contains 2,4 dichlorophenoxyacetic acid 1.5 ~ 2.0mg/L, under dark the cultivation, the enrichment culture callus;
(3) callus that step (2) propagation is grown up is transferred in the MS that contains methyl 0.5 ~ 1.0mg/L and 6-benzyl purine 3.0 ~ 5.0mg/L, and light is cultivated evoking adventive bud;
(4) indefinite bud of step (3) is transferred in the MS that contains methyl 2.5 ~ 3.5mg/L and 6-benzyl purine 2.5 ~ 3.5mg/L, light is cultivated and is induced clove;
(5) clove of step (4) having been opened up leaf is transferred in the MS that contains methyl 1.0 ~ 2.0mg/L, and light is cultivated root induction;
(6) bulb that step (5) has been taken root is transplanted in transplanting medium, and water spray is cultivated; Transplanting medium is that volume ratio is the perlite of 1:1 and the mixed-matrix of humus.
MS is the conventional MS that contains sucrose 30g/L and agar 6g/L.The conventional sucrose that uses is 30g/L, and agar is 6g/L.Light is cultivated to conventional light condition of culture, is specially: illumination 14 ~ 16h/d, intensity of illumination 1200-1300Lux, 25～28 ℃ of temperature.
In the step (l), the concrete operations of getting the embryo of Chinese short-tube lycoris are: with liquid detergent solution washing China short-tube lycoris the surface of the seed, and running water flushing 0.5 ~ 1h, distilled water flushing 3 times is handled seed 30s with 70% alcohol, and aseptic water washing 3 times is about each 30s; Use 0.11%HgCl subsequently
2Soak sterilization 12 ~ 15min, aseptic water washing 4 ~ 6 times, each 30s cuts seed with scalpel, with the embryo in the pincet taking-up seed.
In the step (2), every 20 ~ 25d, the callus lines that propagation is grown up is cut into plurality of small blocks, is transferred to carry out the callus enrichment culture on the proliferated culture medium again.
In the step (4), concentration of sucrose is 40 ~ 60g/L, and the concentration of 6-benzyl purine is 3.0mg/L, and the concentration of methyl is 3.0mg/L.
Beneficial effect: compare with short-tube lycoris tissue culture method of the prior art, the distinguishing feature of the tissue culture quick propagation culturing method of Chinese short-tube lycoris of the present invention is: the present invention is an explant with the embryo of Chinese short-tube lycoris, induce explant to produce callus earlier, and then the generation of evoking adventive bud, clove, finally finish plant regeneration.Therefore the present invention has increased another new way for Chinese short-tube lycoris tissue-culturing quick-propagation; In addition, by the method for evoked callus by way of the generation indefinite bud, not only the growth coefficient height can be produced 1,000,000 cloves in 1 year in this method, can provide effective way for breeding kind of a ball fast; And test-tube plantlet differentiation synchronization extent height, high conformity, the large-scale production exploitation is worth high, has good economic outlook.
Description of drawings
Fig. 1 is the seed of Chinese short-tube lycoris;
Fig. 2 is the embryo of Chinese short-tube lycoris;
Fig. 3 is the callus that Chinese short-tube lycoris embryo induces;
Fig. 4 is the bud of Chinese short-tube lycoris embryo callus differentiation;
Fig. 5 is the clove that grows up to of the bud of Chinese short-tube lycoris embryo callus differentiation bunch;
Fig. 6 is the regeneration plantlet of Chinese short-tube lycoris;
Fig. 7 is the Chinese short-tube lycoris after transplanting.
Embodiment
The present invention is described further below in conjunction with drawings and Examples.
Embodiment 1
MS among this embodiment is the conventional MS that contains sucrose 30g/L and agar 6g/L.
Choose healthy full Chinese short-tube lycoris seed, as shown in Figure 1, with liquid detergent solution washing the surface of the seed, running water flushing 0.5 ~ 1h, distilled water flushing 3 times.Handle seed 30s with 70% alcohol, aseptic water washing 3 times is about each 30s; Use 0.11%HgCl subsequently
2Soak sterilization 12 ~ 15min, aseptic water washing 4 ~ 6 times, each 30s.Cut seed with scalpel, shown in Figure 2 with the embryo in the pincet taking-up seed, it is inoculated into sterilized callus inducing medium MS+2, carry out inducing of callus on 4-D2.0 ~ 3.0mg/L.The callus of induce process is carried out under dark condition, and cultivate about 10d and begin more, and propagation is grown up gradually, as shown in Figure 3, and about the 25th day, callus growth state the best.Be cut into plurality of small blocks with callus lines this moment, transfers in MS+2, on the proliferated culture medium of 4-D1.5 ~ 2.0mg/L, carries out the callus enrichment culture under the dark condition of culture; Every 20 ~ 25 days, the callus lines that propagation is grown up was cut into plurality of small blocks and is transferred on the proliferated culture medium again, carries out the enrichment culture of callus again.Repeat the enrichment culture several times of callus, after reaching predetermined quantity,, be transferred to the last cultivation of medium MS+6-BA3.0 ~ 5.0mg/L+NAA0.5 ~ 1.0mg/L of induced bud differentiation with the high-quality callus of this moment, cultivate about 25d and differentiate indefinite bud, as shown in Figure 4.With the indefinite bud that has differentiated, be transferred to medium MS+6-BA2.5 ~ 3.5mg/L+NAA2.5 ~ 3.5mg/L and cultivate clove, clove bulb diameter reaches about 0.4cm, as shown in Figure 5 behind the cultivation 25d.To open up the clove of leaf, transfer in root media MS+NAA1.0 ~ 2.0mg/L root induction, pH5.8.The growth of the inducing of indefinite bud, clove and take root and all under illumination condition, carry out illumination 14 ~ 16h/d, intensity of illumination 1200 ~ 1300Lux, 24～26 ℃ of temperature.Finish the regeneration plant of taking root and transplant on the mixed-matrix of perlite+humus (1:1), spray water twice every day, promote the seedling growth, wherein, the regeneration plant of taking root as shown in Figure 6, the seedling after the transplanting is as shown in Figure 7.
Embodiment 2
Repeat embodiment 1 by described same steps as, at the induction period of callus, the evoked callus medium is replaced by MS+6-BA0.1 ~ 0.5mg/L+2,4-D1.0 ~ 2.0mg/L+CH250 ~ 500mg/L.Added caseinhydrolysate 250 ~ 500mg/L in the medium, not only improved the healing rate of planting embryo explants, and the callus quality that induces has improved greatly also.
Embodiment 3
Method is with embodiment 1, at the clove cultivation stage, when sucrose concentration in the medium that will induce clove when 30g/L is increased to 40 ~ 60g/L, the diameter of clove just can increase to about 0.7cm from about 0.4cm, promote that obviously bulb expands, play the effect in strong sprout.
Claims (4)
1. the method for quickly breeding of a Chinese short-tube lycoris is characterized in that, may further comprise the steps:
(1) gets the embryo of Chinese short-tube lycoris, be inoculated in inducing culture, the dark evoked callus down of cultivating; Wherein, inducing culture is the MS that contains the MS of 2,4 dichlorophenoxyacetic acid 2.0 ~ 3.0mg/L or contain 6-benzyl purine 0.1 ~ 0.5mg/L, 2,4 dichlorophenoxyacetic acid 1.0 ~ 2.0mg/L, caseinhydrolysate 250 ~ 500mg/L;
(2) callus of step (1) is transferred in the MS that contains 2,4 dichlorophenoxyacetic acid 1.5 ~ 2.0mg/L, under dark the cultivation, the enrichment culture callus;
(3) callus that step (2) propagation is grown up is transferred in the MS that contains methyl 0.5 ~ 1.0mg/L and 6-benzyl purine 3.0 ~ 5.0mg/L, and light is cultivated evoking adventive bud;
(4) indefinite bud of step (3) is transferred in the MS that contains methyl 2.5 ~ 3.5mg/L and 6-benzyl purine 2.5 ~ 3.5mg/L, light is cultivated and is induced clove;
(5) clove of step (4) having been opened up leaf is transferred in the MS that contains methyl 1.0 ~ 2.0mg/L, and light is cultivated root induction;
(6) bulb that step (5) has been taken root is transplanted in transplanting medium, and water spray is cultivated; Transplanting medium is that volume ratio is the perlite of 1:1 and the mixed-matrix of humus;
Wherein, the MS in each step is the conventional MS that contains sucrose and agar.
2. the method for quickly breeding of Chinese short-tube lycoris according to claim 1, it is characterized in that: in the step (l), the concrete operations of getting the embryo of Chinese short-tube lycoris are: with liquid detergent solution washing China short-tube lycoris the surface of the seed, running water flushing 0.5 ~ 1h, distilled water flushing 3 times, handle seed 30s with 70% alcohol, aseptic water washing 3 times is about each 30s; Use 0.11%HgCl subsequently
2Soak sterilization 12 ~ 15min, aseptic water washing 4 ~ 6 times, each 30s cuts seed with scalpel, with the embryo in the pincet taking-up seed.
3. the method for quickly breeding of Chinese short-tube lycoris according to claim 1, it is characterized in that: in the step (2), every 20 ~ 25d, the callus lines that propagation is grown up is cut into plurality of small blocks, is transferred to and carries out the callus enrichment culture on the proliferated culture medium.
4. the method for quickly breeding of Chinese short-tube lycoris according to claim 1, it is characterized in that: in the step (4), the concentration of 6-benzyl purine is 3.0mg/L, and the concentration of methyl is 3.0mg/L, and concentration of sucrose is 40 ~ 60g/L.
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Cited By (12)
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| CN102696488A (en) * | 2012-07-09 | 2012-10-03 | 贵州芊芊园艺新技术发展公司 | Method for rapid propagation of lycoris plant seedlings through hydroponics |
| CN102696479A (en) * | 2012-04-24 | 2012-10-03 | 江苏省中国科学院植物研究所 | Method for propagating stonegarlic quickly and efficiently |
| CN102960243A (en) * | 2012-06-28 | 2013-03-13 | 浙江农林大学 | Method for tissue culture and rapid propagation by basal disc-free scales of Lycoris chinensis |
| CN102972289A (en) * | 2012-06-28 | 2013-03-20 | 浙江农林大学 | Method for tissue culture and rapid propagation by using Lycoris chinensis leaves |
| CN103733871A (en) * | 2014-01-28 | 2014-04-23 | 南京林业大学 | Lycoris longituba cultivation method in composite populus and lycoris longituba cultivation mode |
| CN103782787A (en) * | 2014-02-27 | 2014-05-14 | 湖南中医药大学 | GPA planting method of Lycoris aurea |
| CN103782772A (en) * | 2014-01-28 | 2014-05-14 | 南京林业大学 | Chinese lycoris cultivation method under poplar and Chinese lycoris composite cultivation pattern |
| CN104186320A (en) * | 2014-08-21 | 2014-12-10 | 邓波 | Method for in vitro culture of lycoris longituba seeds |
| CN105475143A (en) * | 2016-01-26 | 2016-04-13 | 广西植物研究所 | Method for obtaining regenerated plant through longtube stonegarlic tissue culture |
| CN108901844A (en) * | 2018-07-09 | 2018-11-30 | 江苏省中国科学院植物研究所 | A method of building Lycoris genetic conversion system |
| CN110235784A (en) * | 2019-07-19 | 2019-09-17 | 东北农业大学 | A method of induction Liao Dong Aralia wood adventitious root occurs and proliferation |
| CN112237142A (en) * | 2020-11-02 | 2021-01-19 | 江苏省中国科学院植物研究所 | Tissue culture medium for lycoris, callus culture method and method for establishing lycoris regeneration system |
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| CN102696479A (en) * | 2012-04-24 | 2012-10-03 | 江苏省中国科学院植物研究所 | Method for propagating stonegarlic quickly and efficiently |
| CN102960243A (en) * | 2012-06-28 | 2013-03-13 | 浙江农林大学 | Method for tissue culture and rapid propagation by basal disc-free scales of Lycoris chinensis |
| CN102972289A (en) * | 2012-06-28 | 2013-03-20 | 浙江农林大学 | Method for tissue culture and rapid propagation by using Lycoris chinensis leaves |
| CN102696488A (en) * | 2012-07-09 | 2012-10-03 | 贵州芊芊园艺新技术发展公司 | Method for rapid propagation of lycoris plant seedlings through hydroponics |
| CN103733871A (en) * | 2014-01-28 | 2014-04-23 | 南京林业大学 | Lycoris longituba cultivation method in composite populus and lycoris longituba cultivation mode |
| CN103782772B (en) * | 2014-01-28 | 2015-08-19 | 南京林业大学 | Lycoris cultivation method under a kind of willow, Lycoris composite cultivation mode |
| CN103782772A (en) * | 2014-01-28 | 2014-05-14 | 南京林业大学 | Chinese lycoris cultivation method under poplar and Chinese lycoris composite cultivation pattern |
| CN103782787B (en) * | 2014-02-27 | 2015-07-22 | 湖南中医药大学 | GAP planting method of Lycoris aurea |
| CN103782787A (en) * | 2014-02-27 | 2014-05-14 | 湖南中医药大学 | GPA planting method of Lycoris aurea |
| CN104186320A (en) * | 2014-08-21 | 2014-12-10 | 邓波 | Method for in vitro culture of lycoris longituba seeds |
| CN104186320B (en) * | 2014-08-21 | 2016-08-24 | 邓波 | A kind of method of Bulbus Lycoridis longitubae seed cultured in vitro |
| CN105475143A (en) * | 2016-01-26 | 2016-04-13 | 广西植物研究所 | Method for obtaining regenerated plant through longtube stonegarlic tissue culture |
| CN108901844A (en) * | 2018-07-09 | 2018-11-30 | 江苏省中国科学院植物研究所 | A method of building Lycoris genetic conversion system |
| CN108901844B (en) * | 2018-07-09 | 2020-01-14 | 江苏省中国科学院植物研究所 | Method for constructing lycoris genus genetic transformation system |
| CN110235784A (en) * | 2019-07-19 | 2019-09-17 | 东北农业大学 | A method of induction Liao Dong Aralia wood adventitious root occurs and proliferation |
| CN112237142A (en) * | 2020-11-02 | 2021-01-19 | 江苏省中国科学院植物研究所 | Tissue culture medium for lycoris, callus culture method and method for establishing lycoris regeneration system |
| CN112237142B (en) * | 2020-11-02 | 2022-04-26 | 江苏省中国科学院植物研究所 | Tissue culture medium for establishing Lycoris chinensis or lycoris aurea regeneration system and method thereof |
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Application publication date: 20111019 Assignee: Jiangsu Jinghua Modern Agricultural Development Co.,Ltd. Assignor: Nanjing Forestry University Contract record no.: X2019320000355 Denomination of invention: Quick propagation method for lycoris chinensis Granted publication date: 20120711 License type: Common License Record date: 20191216 |
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