CN102440185B - Fast culturing method for lily virus-free seed ball - Google Patents
Fast culturing method for lily virus-free seed ball Download PDFInfo
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- CN102440185B CN102440185B CN201010506284.3A CN201010506284A CN102440185B CN 102440185 B CN102440185 B CN 102440185B CN 201010506284 A CN201010506284 A CN 201010506284A CN 102440185 B CN102440185 B CN 102440185B
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Abstract
The invention discloses a comprehensive virus-free technique for manufacturing lily seed balls, namely, thermally treating and planting lily seed balls for 7-15 days at 35-38 degrees centigrade; the stem tip growing points of the thermally treated lily seed balls are taken as explants; vaccinating the explants in a culture medium, of which the Ribavirin content is 5-10mg/L, to be in virus-free cultivation; and detecting the viruses, such as the lily symptomless virus (LSV), the cucumber mosaic virus (CMV) and lily mottle virus (LMoV) of virus-free tissue culture seedling by the enzyme-linked immunosorbent assay test(ELISA) and the RT-PCR (Reverse Transcription-polymerase Chain Reaction) detection; the fast culturing method for the lily virus-free seed ball ensures that the cultured lily seed balls remove the virus infection through the comprehensive virus-free technique; the tissue culture medium component and culture program of the virus-free lily seed balls are optimized; virus-free cultivation seed balls are obtained through fast cultivation; and the fast culturing method belongs to the fields of plant culture rapid propagation technique and plant cell engineering technique.
Description
Technical field
The invention discloses the tissue culture method of flower lily virus-free culture and utilize Sparged bioreactors to expand the cultural method of detoxification clove (kind ball) fast, belonging to the technical field of plant tissue culture fast breeding technique field and plant cell engineering.
Background technology
Lily is important ornamental flower, is mainly used in cutting flower variety and produces.Lily grows under open condition, inevitably by virus infection, lily asymptomatic virus Lily symptomless virus (LSV), cucumber mosaic virus Cucumber mosaic virus (CMV), lily mottle virus Lily mottle virus (LMoV), tulip design of scattered small flowers and plants virus Tulip breaking virus (TBV) and lily rosette virus Lily rosettle virus (LRV) are the common virus affecting lily flower production.Virus infection plant can cause lily economical character serious degradation, greatly have impact on the production of lily.Colonize in the virus in lily bulb, grow up to the process of growth of plant along with bulb eye is sprouted, virus can carry out copying breeding in lily plant body, but does not have virus in the active shoot apical meristem of metabolism.Lily detoxification technology utilizes stem apex part not have virulent characteristic exactly, under gnotobasis condition, utilize the medium of artificial preparation, cultivate virus-free test-tube plantlet or seed ball, then plantlet in vitro and seed ball are isolated artificial or under natural isolation condition, be grown to serve as the commodity kind ball for cut-flower.The consumption figure of recent year lily increases year by year, but during China lily produces at present, the kind ball dependence on import of more than 90% nearly.The production of a small amount of domestic detoxification kind ball, the traditional tissue culture mode of most employing, not only the production cycle is long, but also need to drop into a large amount of manpowers, replaced medium frequently, production process needs very large culture space, in order to ensure culture space temp. and humidity, meeting condition of culture needs and consuming a lot of energy.In order to solve Problems existing in the production of domestic detoxification kind ball, the research of special application bio-reactor fast culture lily detoxification seed ball method.
Summary of the invention
The present invention is with flowers market popular " Siberia Siberia ", " rope side Sorbonne ", " Marco Polo Marco polo ", " Man Nisha Manissa ", " Kang Jiadeao Concad ' 0 " 5 kinds are material, adopt comprehensive detoxification technology by thermal treatment, the method that antiviral agent and Shoot Tip Culture combine, optimize detoxification lily ball group training medium component and cultivation program, and expand detoxification seed ball method fast by bio-reactor liquid culture, shorten fast numerous time of lily detoxification high-quality kind ball, the planting survival rates of seed ball is higher.The present invention is easy to apply in the scale quick propagation of flower lily original seed is produced.The present invention includes: stripping of explant sterilization and growing point; Virus-free culture; The proportioning of each stage medium of group training; Detoxification seed ball in the reactor expand cultivation; Refrigeration vernalization; The several process of rooting culture.
The present invention implements by the following technical programs:
Utilize the method for bio-reactor fast culture lily detoxification kind ball, carry out as follows:
(1) plantation of lily ball: plant lily ball under 35-38 DEG C of condition, through the cultivation of 7-15 days, treats that bud grows tall and bursts forth into rosette-stape, starts to gather lily stem apex;
(2) the stripping of explant sterilization and growing point: the lily stem apex that step (1) is collected, with neutral soap water cleaning 3-5 minute, tap water spends the night, 0.1% mercuric chloride sterilizing 5-10 minute, then in 70% alcohol-pickled 10-15 second, take out stem apex, first rinse 2-3 minute with adding tween aqua sterilisa, 5-6 time is rinsed again with aqua sterilisa, after explant cleans up, first the blade of stem apex is all divested, with dissecting needle picking lily shoot tip meristem, growing point length is 0.4-0.8mm, and the growing point stripped is cultivated explant as group training;
(3) virus-free culture:
1. explant lily shoot tip meristem step (2) stripped, be inoculated on 1/2MS+6-BA 1.0-2.0mg/L+NAA0.2-0.5mg/L+4% sucrose+agar powder 6g/L inducing culture, the pH 5.8 of medium, cultivated through 40-60 days, growing point enlarges into the leafage of diameter 2-3cm;
2. leafage is cut into 0.5cm fritter, be inoculated into MS+NAA0.2-0.5mg/L+4% sucrose+Ribavirin 5-10mg/L+ agar powder 5g/L+0.5% active carbon containing on the medium of virazole, the pH5.8 of medium, cultivated through 40-60 days, formed the bottle seedling of dribbling;
3. Viral diagnosis: by step 2. bottle seedling carry out enzyme linked immunosorbent assay (ELISA) experiment (ELISA) and RT-PCR and detect, confirm without after lily asymptomatic virus (LSV), cucumber mosaic virus (CMV) and lily mottle virus (LMoV) virus;
(4) detoxification seed ball Fiber differentiation: 2. step is transferred to seed ball inducing culture MS+KT2-5mg/L+NAA0.2-0.5mg/L+4% sucrose+agar powder 5g/L+0.5% active carbon through Viral diagnosis virus-free detoxification bottle seedling, the pH 5.8 of medium, temperature 22 ± 1 DEG C, intensity of illumination 1500Lx, cultivate 40-60 days under the environmental condition of illumination 12h/ days, directly induce band leaf detoxification seed ball;
(5) Multiplying culture: by step (4) band leaf detoxification seed ball, cut blade and base thereof tissue, rip cutting becomes 3-4 block diameter 0.3-0.5cm material, be inoculated on the medium identical with detoxification seed ball Fiber differentiation, temperature 22 ± 1 DEG C, cultivate two months under the environmental condition of illumination 12h/ days, intensity of illumination 1000-1500Lx, numerous 3-4 can be expanded doubly;
(6) detoxification seed ball expand cultivation: detoxification seed ball step (5) cultivated separately, forms single small seed ball, diameter 0.3-0.5cm, is seeded in liquid culture in bio-reactor; Liquid nutrient medium MS+NAA0.2-0.5mg/L+ forchlorfenuron CPPU1-4mg/L+6% sucrose, the pH 5.8 of medium, condition of culture, temperature 22 ± 1 DEG C, intensity of illumination 1500Lx and 2L/min throughput, cultivate 45 days, cultivate seed bulb diameter and can reach 1.0-1.4cm, root system is at 3-5 bar/grain, and seed ball weighs 0.7-1.2g/ grain;
(7) refrigerate: take out the detoxification seed ball that step (6) is cultivated, rinse out culture fluid with clear water, be wrapped in the sterile peat composed of rotten mosses, through 3-4 DEG C of chilling treatment 30-40 days breaking dormancy;
(8) domestication is transplanted: taken out by step (7) seed ball, reach liquid with 2000 times of Amicis and soak seed ball 20 minutes, then field planting is in the cave dish in 80-90 cave, plug media: sabot mixed thoroughly by the bacterial manure that the peat composed of rotten mosses 1 part, vermiculite 1 part add a small amount of growth-promoting root.Water permeable after field planting, and with plastic mulching cave dish, be put into maintenance in solarium, daytime temperature 20-25 DEG C, night temperature 10-15 DEG C, dish soil maintenance moistening, after field planting survives, weekly spray a 1/2MS nutrient solution;
(9) treat that seed bulb diameter grows to more than 2cm, select tool Cold and cool climate in summer 800 ~ 1000 meters of Altitude Regions, taking shelter from rain with under insect protected isolation condition, late March is to field planting mid-April in seedbed.
Claims (2)
1. utilize the method for bio-reactor fast culture lily detoxification kind ball, carry out as follows:
(1) the stripping of explant sterilization and growing point: plant lily ball under 35-38 DEG C of condition, through the cultivation of 7-15 days, treat that bud grows tall and bursts forth into rosette-stape, start to gather lily stem apex, with neutral soap water cleaning 3-5 minute, tap water spends the night, 0.1% mercuric chloride sterilizing 5-10 minute, then in 70% alcohol-pickled 10-15 second, take out stem apex, first rinse 2-3 minute with adding tween aqua sterilisa, 5-6 time is rinsed again with aqua sterilisa, after explant cleans up, start to strip growing point, first the blade of stem apex is all divested, with dissecting needle picking lily shoot tip meristem, growing point length is 0.4-0.8mm, the growing point stripped is inoculated immediately,
(2) virus-free culture: step is Fiber differentiation 1., the lily shoot tip meristem stripped is inoculated into inducing culture 1/2MS+6-BA1.0-2.0mg/L+NAA0.2-0.5mg/L+4% sucrose+agar powder 6g/L, the pH 5.8 of medium, cultivated through 40-60 days, growing point enlarges into the leafage of diameter 2-3cm; 2. leafage is cut into 0.5cm fritter, is inoculated in the medium MS+NAA0.2-0.5mg/L+4% sucrose+Ribavirin5-10mg/L+ agar powder 5g/L+0.5% active carbon containing virazole, the pH 5.8 of medium, cultivated through 40-60 days, form the bottle seedling of dribbling;
(3) Viral diagnosis: by step 2. bottle seedling deliver to flowers product quality supervision and inspection test center of the Ministry of Agriculture, confirm after testing not to be with lily asymptomatic virus, cucumber mosaic virus and lily mottle virus without any damage by disease and insect phenomenon, be just confirmed to be lily detoxification seedling;
(4) detoxification seed ball Fiber differentiation: through Viral diagnosis, by step 2. detoxification bottle seedling transfer to seed ball inducing culture MS+KT2-5mg/L+NAA0.2-0.5mg/L+4% sucrose+agar powder 5g/L+0.5% active carbon, the pH 5.8 of medium, temperature 22 ± 1 DEG C, intensity of illumination 1500Lx, cultivate 40-60 days under the environmental condition of illumination 12h/ days, directly induce band leaf detoxification seed ball;
(5) Multiplying culture: by step (4) band leaf detoxification seed ball, after cutting blade and base thereof tissue, rip cutting becomes 3-4 block diameter 0.3-0.5cm material, be inoculated on the medium identical with seed ball Fiber differentiation, temperature 22 ± 1 DEG C, cultivate two months under the environmental condition of illumination 12h/ days, intensity of illumination 1000-1500Lx, expand numerous 3-4 doubly;
(6) detoxification seed ball liquid culture in bio-reactor: detoxification seed ball step (5) cultivated separately, forms single small seed ball, diameter 0.3-0.5cm, is seeded in liquid culture in bio-reactor; Liquid nutrient medium MS+NAA 0.2-0.5mg/L+ forchlorfenuron CPPU1-4mg/L+6% sucrose, the pH 5.8 of medium, condition of culture temperature 22 ± 1 DEG C, intensity of illumination 1500Lx and 2L/min throughput, cultivate 45 days, cultivate detoxification seed bulb diameter and reach 1.0-1.4cm, root system is at 3-5 bar/grain, and seed ball weighs 0.7-1.2g/ grain;
(7) refrigerate: take out the detoxification seed ball that step (6) is cultivated, rinse out culture fluid with clear water, be wrapped in the sterile peat composed of rotten mosses, through 3-4 DEG C of chilling treatment 30-40 days breaking dormancy;
(8) domestication is transplanted: taken out by step (7) seed ball, reach liquid with 2000 times of Amicis and soak seed ball 20 minutes, then field planting is in the cave dish in 80-90 cave, plug media: sabot mixed thoroughly by the bacterial manure that the peat composed of rotten mosses 1 part, vermiculite 1 part add a small amount of growth-promoting root, water permeable after field planting, and with plastic mulching cave dish, be put into maintenance in solarium, daytime temperature 20-25 DEG C, night temperature 10-15 DEG C, dish soil keeps moistening, after field planting survives, sprays a 1/2MS nutrient solution weekly;
(9) treat that detoxification seed bulb diameter grows to more than 2cm, select tool Cold and cool climate in summer 800 ~ 1000 meters of Altitude Regions, taking shelter from rain with under insect protected isolation condition, late March is to field planting mid-April in seedbed.
2. method according to claim 1, its feature is that culture materials is " Siberia Siberia ", " rope side Sorbonne ", " Marco Polo Marco polo ", " Man Nisha Manissa ", " Kang Jiadeao Concad ' O " 5 kinds.
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| CN103283604B (en) * | 2013-06-07 | 2015-02-18 | 南京工业大学大丰海洋产业研究院 | Method for removing endophytic bacteria of explants in lily tissue culture |
| CN105613283A (en) * | 2014-10-27 | 2016-06-01 | 松桃宏发肉食品有限责任公司 | Fast culturing method for lily virus-free bulblets |
| CN106718931B (en) * | 2017-01-19 | 2019-07-23 | 重庆市风景园林科学研究院 | The method for carrying out succulent breeding using bioreactor |
| CN109042334A (en) * | 2018-09-21 | 2018-12-21 | 杨晓峰 | A kind of Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait detoxification tissue cultures and rapid propagation method |
| CN110574684A (en) * | 2019-10-08 | 2019-12-17 | 井冈山大学 | rapid breeding method of longya lily bulbs |
| CN112243631B (en) * | 2020-09-14 | 2022-03-22 | 云南省农业科学院花卉研究所 | Method for rapidly breaking dormancy of green flower lily seed bulbs |
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