CN105613283A

CN105613283A - Fast culturing method for lily virus-free bulblets

Info

Publication number: CN105613283A
Application number: CN201410581640.6A
Authority: CN
Inventors: 杨长能
Original assignee: Songtao Hongfa Meat Food Co Ltd
Current assignee: Songtao Hongfa Meat Food Co Ltd
Priority date: 2014-10-27
Filing date: 2014-10-27
Publication date: 2016-06-01

Abstract

The invention discloses a fast culturing method for lily virus-free bulblets; the fast culturing method comprises the steps: lily bulblets are thermally treated and planted for 7-15 days under a condition of the temperature of 35-38 DEG C; the stem tip growing points of the thermally treated lily bulblets are peeled to be taken as explants; a culture medium with the ribavirin content of 5-10 mg/L is inoculated with the explants, and virus-free cultivation is performed; and viruses, such as lily symptomless virus (LSV), cucumber mosaic virus (CMV) and lily mottle virus (LMoV) of virus-free tissue culture seedlings are detected by an enzyme-linked immunosorbent assay test (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) detection. The fast culturing method for the lily virus-free bulblets ensures that the cultured lily bulblets have the virus infection removed through the comprehensive virus-free technique; the tissue culture medium components and culture program of the virus-free lily bulblets are optimized; the virus-free tissue culture bulblets are obtained through fast culture; and the fast culturing method belongs to the technical field of plant tissue culture rapid propagation and the technical field of plant cell engineering.

Description

A kind of fast culturing method for lily virus-free seed ball

Technical field

The invention discloses the tissue culture method of flower lily virus-free culture and utilize Sparged bioreactors quickly to expand the cultural method of detoxification clove (kind ball), belonging to the technical field of plant tissue culture fast breeding technique field and plant cell engineering.

Background technology

Bulbus Lilii is important ornamental flower, is mainly used in cutting flower variety and produces. Bulbus Lilii grows under open condition, inevitably by virus infection, lily asymptomatic virus Lilysymptomlessvirus (LSV), cucumber mosaic virus Cucumbermosaicvirus (CMV), lily mottle virus Lilymottlevirus (LMoV), Flos Tulipae Gesnerianae design of scattered small flowers and plants virus Tulipbreakingvirus (TBV) and lily rosette virus Lilyrosettlevirus (LRV) affect the common virus that lily flower produces. Virus infection plant can cause Bulbus Lilii economical character serious degradation, greatly have impact on the production of Bulbus Lilii. Colonizing in the virus in lily bulb, along with the sprouting of bulb eye grows up to the growth course of plant, virus can carry out replicating breeding in Bulbus Lilii plant body, but does not have virus in the shoot apical meristem that metabolism enlivens. Lily detoxification technology utilizes stem apex part not have virulent characteristic exactly, when gnotobasis, utilize the culture medium of human configuration, cultivate virus-free test tube Seedling or seed ball, then by tissue cultured seedling and seed ball being manually isolated or when natural isolation, it is grown to serve as the commodity kind ball for cut-flower. The consumption figure of recent year Bulbus Lilii increases year by year, but during China Bulbus Lilii 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, most employing tradition tissue culture modes, not only the production cycle is long, but also need to put into substantial amounts of manpower, change culture medium frequently, production process needs very big culture space, in order to ensure culture space humiture, meets condition of culture needs and consumes a lot of energy. In order to solve Problems existing during domestic detoxification kind ball produces, the research of special application bioreactor fast culture lily detoxification seed ball method.

Summary of the invention

The present invention includes: stripping of outer implant sterilizing and growing point; Virus-free culture; Group trains the proportioning of each stage culture medium; Detoxification seed ball in the reactor expand cultivation; Cold preservation vernalization; The several process of rooting culture. The present invention selects flowers market popular " Siberia " (Siberia), " rope side " (Sorbonne), " Marco Polo " (Marcopolo), " Man Nisha " (Manissa), " Kang Jiadeao " (Concad'0) 5 kinds are material, adopt comprehensive detoxification technology, by the method that heat treatment, antiviral agent and Shoot Tip Culture combine. Optimizing detoxification lily ball group training medium component and cultivation program, fast culture obtains detoxication and tissue culture seed ball, shortens fast numerous time of lily detoxification high-quality kind ball, and the planting survival rates of seed ball is higher simultaneously. The present invention is prone to popularization and application in the scale quick propagation of flower lily original seed produces.

The present invention is achieved through the following technical solutions:

A kind of fast culturing method for lily virus-free seed ball method, carries 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 after plantation, treats that bud grows tall the middle part likeness in form rosette-stape of the raw stem of leaf collection.

(2) the stripping of outer implant sterilizing and growing point: the Bulbus Lilii stem apex of acquisition step (1), cleans 3-5 minute with neutral soap water, and overnight, 0.1% mercuric chloride sterilizing 5-10 minute, then in 70% alcohol-pickled 10-15 second for tap water. Take out stem apex, first with adding tween aquesterilisa flushing 2-3 minute, then rinse 5-6 time with aquesterilisa. After outer implant cleans up, starting to strip growing point, first all divested by the blade of stem apex, with dissecting needle picking Bulbus Lilii shoot tip meristem, growing point length is 0.4-0.8 (mm), and as group training, the growing point stripped is cultivated outer implant.

(3) virus-free culture: outer implant Bulbus Lilii shoot tip meristem step (3) 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 (pH5.8)), cultivating through 40-60 days, growing point enlarges into the leafage of diameter 2-3cm. 2. leafage is cut into 0.5cm fritter, it is inoculated in the culture medium (MS+NAAO.2-0.5mg/L+4% sucrose+virazole (Ribavirin) 5-l0mg/L+ agar powder 5g/L+O.5% activated carbon (pH5.8)) containing virazole, cultivated through 40-60 days, form the bottle Seedling of dribbling.

(4) Viral diagnosis: step 2. bottle Seedling carries out enzyme-linked immunosorbent assay experiment (ELISA) and RT-PCR detection, confirms without lily asymptomatic virus (LSV), cucumber mosaic virus (CMV) and lily mottle virus (LMoV) virus.

(5) detoxification seed ball inducing culture: 2. step is transferred to seed ball inducing culture (MS+KT2-5mg/L+NAA0.2-0.5mg/L+4% sucrose+agar powder 5g/L+O.5% activated carbon (pH5.8)) through the virus-free detoxification bottle Seedling of Viral diagnosis, temperature 22 scholar 1 DEG C, intensity of illumination 1500Lx, cultivate 40-60d under the environmental condition of illumination 12h/d, directly induce detoxification seed ball.

(6) enrichment culture: by step (4) group training seed ball, after cutting blade and base thereof tissue, rip cutting becomes 3-4 block diameter 0.3-0.5cm material, it is inoculated in the culture medium identical with seed ball inducing culture, temperature 22 scholar 1 DEG C, cultivate 2 months under the environmental condition of illumination 12h/d, intensity of illumination 1000-1500Lx, it is possible to expanding propagation 3-4 times.

(7) detoxification seed ball expand cultivation: group training seed ball step (5) cultivated is separately, form single small seed ball, about diameter 0.3-0.5cm, it is inoculated in seed ball and expands culture medium (MS+NAA0.2-0.5mg/L+ forchlorfenuron CPPU1-4mg/L+6% sucrose (pH5.8)), cultivate 45 days under 22 scholar 1 DEG C, 1500Lx12h illumination, 12h dark condition, cultivate seed bulb diameter up to 1.0-1.4cm, root system is at 3-5 bar/grain, seed ball weight 0.7-1.2g/ grain.

(8) cold preservation: take out the detoxification seed ball that step (6) is cultivated, rinse out culture fluid with clear water, is wrapped in the sterilization peat composed of rotten mosses, through 30-40 days breaking dormancies of 3-4 DEG C of chilling treatment.

(9) domestication is transplanted: taken out by step (7) seed ball, reach medicinal liquid with 2000 times of Amicis and soak seed ball 20 minutes, then field planting is in the hole tray in 80-90 cave, plug media: the peat composed of rotten mosses 1 part, Vermiculitum 1 part add the bacterial manure of a small amount of growth-promoting root and mix sabot thoroughly. Water permeable after field planting, and use covering with ground sheeting hole tray. Being put in solarium maintenance, daytime temperature 20-25 DEG C, night temperature 10-15 DEG C, dish soil keeps moistening, after field planting survives, and 1/2MS nutritional solution of spray weekly.

(10) treat that seed bulb diameter grows to more than 2cm, 800��1000 meters of Altitude Regions of optional tool Cold and cool climate in summer, taking shelter from rain and when insect protected isolation, late March to field planting mid-April to seedbed in.

Claims

1. a fast culturing method for lily virus-free seed ball, it is characterised in that carry out as follows:

(1) the stripping of outer implant sterilizing 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 Bulbus Lilii stem apex, clean 3-5 minute with neutral soap water, overnight, 0.1% mercuric chloride sterilizing 5-10 minute, then in 70% alcohol-pickled 10-15 second for tap water; Take out stem apex, first with adding tween aquesterilisa flushing 2-3 minute, then rinse 5-6 time with aquesterilisa; After outer implant cleans up, starting to strip growing point, first all divested by the blade of stem apex, with dissecting needle picking Bulbus Lilii shoot tip meristem, growing point length is 0.4-0.8 (mm), is inoculated immediately by the growing point stripped;

(2) virus-free culture: step is inducing culture 1., the Bulbus Lilii 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 (pH5.8)), cultivating through 40-60 days, growing point enlarges into the leafage of diameter 2-3 (cm);

2. leafage is cut into 0.5 (em) fritter, it is inoculated in the culture medium (MS+NAA0.2-0.5mg/L+4% sucrose+virazole (Ribavirin) 5-10mg/L+ agar powder 5g/L+0.5% activated carbon (pH5.8)) containing virazole, cultivated through 40-60 days, form the bottle Seedling of dribbling;

(3) Viral diagnosis: step 2. bottle Seedling is delivered to, flowers product quality supervision and inspection test center of the Ministry of Agriculture (Kunming), confirm without any pest and disease damage phenomenon and without viruses such as lily asymptomatic virus (LSV), cucumber mosaic virus (CMV) and lily mottle virus (LMoV) after testing, just confirm it is lily detoxification seedling;

(4) detoxification seed ball inducing culture: through Viral diagnosis, step 2. detoxification bottle Seedling is transferred to seed ball inducing culture (MS+KT2-5mg/L+NAA0.2-0.5mg/L+4% sucrose+agar powder 5g/L+0.5% activated carbon (pH5.8)), temperature 22 �� 1 DEG C, intensity of illumination 1500Lx, cultivate 40-60d under the environmental condition of illumination 12h/d, directly induce detoxification seed ball;

(5) enrichment culture: by step (4) group training seed ball, after cutting blade and base thereof tissue, rip cutting becomes 3-4 block diameter 0.3-0.5cm material, it is inoculated in the culture medium identical with seed ball inducing culture, temperature 22 �� 1 DEG C, cultivate 2 months under the environmental condition of illumination 12h/d, intensity of illumination 1000-1500Lx, it is possible to expanding propagation 3-4 times;

(6) detoxification seed ball liquid culture in bioreactor: group training seed ball step (5) cultivated separately, forms single small seed ball, about diameter 0.3-0.5cm, is then inoculated in reactor; Fluid medium (MS+NAA0.2-0.5mg/L+ forchlorfenuron CPPU1-4mg/L+6% sucrose (pH5.8)), 22 �� 1 DEG C, 1500Lx and 2L/min ventilation when cultivate 45 days, cultivate seed bulb diameter up to 1.0-1.4cm, root system is at 3-5 bar/grain, seed ball weight 0.7-1.2g/ grain;

(7) cold preservation: take out the detoxification seed ball that step (6) is cultivated, rinse out culture fluid with clear water, is wrapped in the sterilization peat composed of rotten mosses, through 30-40 days breaking dormancies of 3-4 DEG C of chilling treatment;

(8) domestication is transplanted: taken out by step (7) seed ball, reach medicinal liquid with 2000 times of Amicis and soak seed ball 20 minutes, then field planting is in the cave label in 80-90 cave, plug media: the peat composed of rotten mosses 1 part, Vermiculitum 1 part add the bacterial manure of a small amount of growth-promoting root and mix sabot thoroughly; Water permeable after field planting, and use covering with ground sheeting hole tray; Being put in solarium maintenance, daytime temperature 20-25 DEG C, night temperature 10-15 DEG C, dish soil keeps moistening, after field planting survives, and 1/2MS nutritional solution of spray weekly;

(9) treat that seed bulb diameter grows to more than 2cm, 800��1000 meters of Altitude Regions of optional tool Cold and cool climate in summer, taking shelter from rain and when insect protected isolation, late March to field planting mid-April to seedbed in.