CN106993534B - A method of preventing Chinese rose tissue-cultured seedling browning - Google Patents
A method of preventing Chinese rose tissue-cultured seedling browning Download PDFInfo
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- CN106993534B CN106993534B CN201710277469.3A CN201710277469A CN106993534B CN 106993534 B CN106993534 B CN 106993534B CN 201710277469 A CN201710277469 A CN 201710277469A CN 106993534 B CN106993534 B CN 106993534B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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Abstract
The present invention relates to a kind of methods for preventing Chinese rose tissue-cultured seedling browning, and this method comprises the following steps: 1), drawing materials；2) it, sterilizes: 3), before inoculation pre-processing: the explant sterilized before inoculation being impregnated 10 minutes or so with the aqueous povidone solution (concentration 2-3g/L) for having filtered disinfection and is pre-processed；4), inoculation, Fiber differentiation: pretreated Stem Sections in Rosa Chinensis Jacq is paved and is inoculated in induced medium after being dried on the filter paper sterilized, until adventitious bud is long to 3-4cm, to Multiplying culture, 5), Multiplying culture: the adventitious bud of inductive formation is cut, it is cultivated in proliferated culture medium, subculture is primary within 15-20 days, 6), culture of rootage: the tissue-cultured seedling of Multiplying culture is cut into single plant and is inoculated in root media culture.Present invention optimizes the operating method in Chinese rose tissue cultures each stage, melting brown rate is effectively reduced to 10% hereinafter, solving the problems, such as that Chinese rose tissue culture melting brown rate is high.
Description
Technical field
The present invention relates to a kind of anti-browning methods of Plant Tissue Breeding, and in particular to a kind of Chinese rose tissue-cultured seedling anti-browning
Method.
Background technique
Chinese rose is one of ten great tradition famous flower of China, is known as spending middle queen, also known as Chinese rose.Chinese rose is in afforestation
There is irreplaceable value, the four seasons equal ornamental, the florescence is long, and ornamental value is high, very extensive in the landscape application of north and south.
The propagation method of Chinese rose has traditional cuttage and grafting, but is difficult to a large amount of quickly breedings, later period hair because being limited to propagation material
The tissue cultures of exhibition can breed a large amount of excellent nursery stock in a short time, become quick breeding maximally efficient in Chinese rose breeding
One of means.For woody plant tissure culture, browning is to influence the successful crucial problem of tissue culture.Browning is due to explant
Body makes culture medium browning after the oxidation of phenolic substances that notch releases, therewith explant also can browning, and then lead to its death.
The present invention problem serious for the browning of Chinese rose tissue-cultured seedling, proposes a kind of tissue cultures side for preventing Chinese rose browning
Method, the present invention is by control Chinese rose materials, induction, proliferation, the ingredient of root media, hormone combination, pH and explant
Pre-treatment etc. can effectively prevent phenolic substances to aoxidize, to realize the generation for preventing Chinese rose browning, Chinese rose is greatly improved
Quick reproductive efficiency.
Summary of the invention
The object of the present invention is to provide a kind of methods for preventing Chinese rose tissue-cultured seedling browning, and Chinese rose is greatly improved and quickly breeds effect
Rate.
The technical solution adopted by the present invention are as follows:
A method of Chinese rose tissue-cultured seedling browning is prevented, is included the following steps:
1) it, draws materials: choosing the semi-lignified Chinese rose stem with bud for being in vigorous growth state；It removes stem apex and top children is tender
2-3cm stem-segment with single bud is cut into after stem section；
2) it, sterilizes:
3), pretreatment before inoculation: by the polyvinylpyrrolidone for having filtered disinfection of the explant sterilized before inoculation
Aqueous solution (concentration 2-3g/L), which impregnates 10 minutes or so, to be pre-processed；
4), inoculation, Fiber differentiation: pretreated Stem Sections in Rosa Chinensis Jacq is paved and is inoculated in after being dried on the filter paper sterilized
In induced medium, until adventitious bud is long to 3-4cm, to Multiplying culture, inducing culturing condition is 22 DEG C/daytime, 20 DEG C/black
Night, 12h/ daytime, 12h/ night, intensity of illumination 3000-4000Lux；
5), Multiplying culture: the adventitious bud of inductive formation being cut, is cultivated in proliferated culture medium, 15-20 days subcultures
Once, Multiplying culture condition is 22 DEG C/daytime, 20 DEG C/night, 12h/ daytime, and 12h/ night, intensity of illumination 3000-
4000Lux；
6), culture of rootage: being cut into single plant for the tissue-cultured seedling of Multiplying culture and be inoculated in root media, 20 DEG C/daytime, 18
DEG C/night, 16h/ daytime, at 8h/ night, intensity of illumination is cultivated under the conditions of being 4000-5000Lux.
Preferably, the step 2) sterilisation step are as follows: 1-2h is rinsed with flowing running water, after 75% alcohol impregnates 30s
Sterile purified water flushing 3-5 times, disinfection are used with sterilized distilled water flushing 3-5 times, then after handling 15min with 5%NaClO
Period constantly rock make its disinfection sufficiently, excision sterilized stem section head and the tail be placed in sterilized filter paper dry it is rear to be seeded.
Preferably, the hormone and its content added in induced medium is respectively as follows: 6-BA 0.5-1mg/L, NAA 0.01-
0.1mg/L, the hormone and its content added in proliferated culture medium be respectively as follows: 6-BA 0.5-1mg/L, NAA 0.01-0.1mg/L,
GA30.01mg/L, the hormone and its content added in root media are respectively as follows: NAA 0.2-0.4mg/L, IBA0.1-
0.2mg/L。
Preferably, induced medium, proliferated culture medium, agar consumption is 7.5-8g/L in root media.
Preferably, induced medium, proliferated culture medium, root media pH value be adjusted to 5.5-5.8.
Preferably, active carbon, activated carbon dosage 0.5-1g/L are added in proliferated culture medium, root media.
Preferably, the formula of induced medium are as follows: MS+30g/L sucrose+7.5-8g/L agar+0.5-1mg/L 6- benzyl ammonia
Base purine+0.01-0.1mg/L methyl α-naphthyl acetate, pH5.5-5.8.The formula of proliferated culture medium are as follows: MS+30g/L sucrose+7.5-8g/L
Agar+0.5-1mg/L 6-benzyl aminopurine+0.01-0.1mg/L methyl α-naphthyl acetate+0.01mg/L gibberellin+0.5-1g/L active carbon,
pH5.5-5.8.The formula of root media are as follows: 1/2MS+25g/L sucrose+7.5-8g/L agar+0.2-0.4mg/L methyl α-naphthyl acetate+
0.1-0.2mg/L indolebutyric acid+0.5-1g/L active carbon, pH 5.5-5.8.
Possessed by of the invention the utility model has the advantages that
Present invention optimizes the operating method in Chinese rose tissue cultures each stage, melting brown rate is effectively reduced to 10% hereinafter,
Solve the problems, such as that Chinese rose tissue culture melting brown rate is high.Specifically:
Present invention employs two kinds of adsorbents of polyvinylpyrrolidone and active carbon, are impregnated and are made using polyvinylpyrrolidone
For the pre-treating method of explant, shape after phenolic substances and oxidation of the addition active carbon to adsorb explant release in culture medium
At quinones substance, to achieve the effect that pre- anti-browning.
The further preferred culture medium prescription in Chinese rose tissue cultures each stages of the present invention, effectively reduces Chinese rose tissue culture
Browning problem in the process.
The hardness of culture medium will affect the diffusion of phenolic substances, and the appropriate agar consumption that increases can reduce melting brown rate, the present invention
It is preferred that agar consumption is 7.5-8g/L.
The low pH of culture medium has the function of reduction polyphenol oxidase activity, is able to suppress browning；Culture medium of the present invention
PH value is adjusted to 5.5-5.8.
The phenolic substances of Chinese rose explant wound accumulation can cause browning for a long time, can be with by shortening subculture cycle
Mitigate the browning of Chinese rose tissue-cultured seedling.The present invention shortens to the squamous subculture time within 20 days.
Temperature directly affects enzymatic activity in plant, and temperature is suitably reduced under the premise of guaranteeing plant normal growth can be with
Related enzyme activity is reduced, to inhibit the synthesis of phenolic substances, and then mitigates the generation of browning.Chinese rose is proliferated by the present invention
Culture is placed in 22 DEG C/daytime, is cultivated under the conditions of 20 DEG C/night；Culture of rootage is placed in 20 DEG C/daytime, is trained under the conditions of 18 DEG C/night
It supports.
Specific embodiment
The present invention will be further described combined with specific embodiments below, but the scope of protection of the present invention is not limited.
A method of Chinese rose tissue-cultured seedling browning is prevented, is included the following steps:
The Chinese rose stem with bud of northern area late spring season semi-lignified is chosen, is cut after removing stem apex and top tender stem segments
At 2-3cm stem-segment with single bud, flowing running water rinses 1h, with sterilized distilled water flushing 3 times, 5% after 75% ethanol postincubation 30s
It is rinsed 3 times after NaClO processing 15min with sterile purified water and is carried out disinfection to explant, constantly rocking during disinfection makes it disappear
It is malicious sufficiently, excision sterilized stem section head and the tail be placed in sterilized filter paper dry it is rear to be seeded.
It is that 0.22 μm of filter membrane filters 2g/L aqueous povidone solution with aperture, Stem Sections in Rosa Chinensis Jacq to be seeded is soaked
The 10min in above-mentioned sterilized polyvinylpyrrolidone is steeped, paves and is inoculated in induction training after drying on the filter paper sterilized
It supports in base (MS+30g/L sucrose+7.5g/L agar+0.5mg/L 6-benzyl aminopurine+0.01mg/L methyl α-naphthyl acetate, pH5.8), lures
Leading condition of culture is 22 DEG C/daytime, 20 DEG C/night, 12h/ daytime, and 12h/ night, intensity of illumination 3300Lux.
When the adventitious bud through Fiber differentiation it is long to 4-5cm when, the adventitious bud of inductive formation is cut, in proliferated culture medium
(MS+30g/L sucrose+7.5g/L agar+0.5mg/L 6-benzyl aminopurine+0.01mg/L methyl α-naphthyl acetate+0.01mg/L gibberellin+
0.5g/L active carbon, pH5.6) in cultivated, subculture is primary within 15 days, Multiplying culture condition be 22 DEG C/daytime, 20 DEG C/night,
12h/ daytime, 12h/ night, intensity of illumination 3300Lux.
After Chinese rose tissue-cultured seedling culture to 5-6cm, be cut into simple bud be placed in root media (1/2MS+25g/L sucrose+
7.5g/L agar+0.2mg/L methyl α-naphthyl acetate+0.1mg/L indolebutyric acid+0.5g/L active carbon, pH5.6) in, 20 DEG C/daytime, 18
DEG C/night, 16h/ daytime, 8h/ night cultivates under the conditions of intensity of illumination 4500Lux.
This method efficiently solves the problems, such as that Chinese rose tissue cultures melting brown rate is high, improves each growth phase of Chinese rose tissue-cultured seedling
Chinese rose tissue-cultured seedling melting brown rate is effectively dropped to 10% hereinafter, significant to Chinese rose industrialization production by committed step.
Claims (1)
1. a kind of method for preventing Chinese rose tissue-cultured seedling browning, it is characterised in that: step are as follows:
The Chinese rose stem with bud of northern area late spring season semi-lignified is chosen, is cut into 2- after removing stem apex and top tender stem segments
3 cm stem-segment with single bud, flowing running water rinse 1h, with sterilized distilled water flushing 3 times after 75% ethanol postincubation 30s, 5%NaClO
It is rinsed 3 times after processing 15min with sterile purified water and is carried out disinfection to explant, constantly rocking during disinfection fills its disinfection
Point, excision sterilized stem section head and the tail be placed in sterilized filter paper dry it is rear to be seeded；
It is that 0.22 μm of filter membrane filters 2g/L aqueous povidone solution with aperture, Stem Sections in Rosa Chinensis Jacq to be seeded is soaked in
10min in above-mentioned sterilized polyvinylpyrrolidone paves and is inoculated in induced medium after drying on the filter paper sterilized
In, the induced medium composition are as follows: MS+30g/L sucrose+7.5g/L agar+0.5mg/L 6-benzyl aminopurine+0.01mg/L
Methyl α-naphthyl acetate, pH5.8, inducing culturing condition are 22 DEG C/daytime, and 20 DEG C/night, 12h/ daytime, at 12h/ night, intensity of illumination is
3300Lux；
When the adventitious bud through Fiber differentiation it is long to 4-5cm when, the adventitious bud of inductive formation is cut, is carried out in proliferated culture medium
Culture, the proliferated culture medium composition are as follows: MS+30g/L sucrose+7.5g/L agar+0.5mg/L 6-benzyl aminopurine+
0.01mg/L methyl α-naphthyl acetate+0.01mg/L gibberellin+0.5g/L active carbon, pH5.6, subculture is primary within 15 days, and Multiplying culture condition is
22 DEG C/daytime, 20 DEG C/night, 12h/ daytime, 12h/ night, intensity of illumination 3300Lux；
It after Chinese rose tissue-cultured seedling culture to 5-6cm, is cut into simple bud and is placed in root media, the root media group becomes 1/
2 MS+25g/L sucrose+7.5g/L agar+0.2mg/L methyl α-naphthyl acetate+0.1mg/L indolebutyric acid+0.5g/L active carbons, pH5.6,
20 DEG C/daytime, 18 DEG C/night, 16h/ daytime, 8h/ night is cultivated under the conditions of intensity of illumination 4500Lux.
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| JPH0642809B2 (en) * | 1989-04-04 | 1994-06-08 | 新王子製紙株式会社 | How to make shoot base lumps |
| JPH09121847A (en) * | 1995-10-27 | 1997-05-13 | Nippon Oil Co Ltd | Culture of plant cell |
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| CN106106180A (en) * | 2016-08-04 | 2016-11-16 | 贵州省植物园 | The micropropagation method of Chinese rose of cut flower " Corolla " |
| CN106489737A (en) * | 2016-11-04 | 2017-03-15 | 河南红枫种苗股份有限公司 | A kind of culture medium of Hybrid Tea tissue cultures and method |
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