CN110810241A - Tissue culture seedling propagation method for raspberries - Google Patents
Tissue culture seedling propagation method for raspberries Download PDFInfo
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- CN110810241A CN110810241A CN201911161594.3A CN201911161594A CN110810241A CN 110810241 A CN110810241 A CN 110810241A CN 201911161594 A CN201911161594 A CN 201911161594A CN 110810241 A CN110810241 A CN 110810241A
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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 invention discloses a tissue culture seedling propagation method of raspberries, belongs to the technical field of plant tissue culture, and aims to provide a tissue culture seedling propagation method of raspberries, which has the advantages of short propagation period, high efficiency and low cost, and the technical scheme is characterized by comprising the following steps: s1: selecting an explant; s2: sterilizing explants; s3: performing induction culture; s4: carrying out proliferation culture; s5: and (5) rooting culture. According to the invention, through a plant tissue culture technology, buds are used as raw materials, the method is convenient to obtain, the multiplication rate is up to 6-8 times, the rooting rate reaches 95%, the seedling size is neat, the characters are stable, the cost is saved, the technical support is provided for industrial production of raspberries, and the method has an important significance for large-scale production of high-quality raspberries seedlings by enterprises.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a tissue culture seedling propagation method of raspberries.
Background
Raspberry (Rubus idaeus) is a berry fruit tree of Rubus of rosaceae and is known as one of four berries in the world. The fruit is rich in nutrition, sour, sweet, tasty and rich in nutrition, and can be eaten fresh or processed. Has the functions of resisting oxidation, regulating metabolism, resisting tumor, preventing cardiovascular and cerebrovascular diseases, enhancing immunity and the like, and has great application and development values. The raspberry has a long history of cultivation in Europe and America, the introduction of the raspberry has more than 30 years history in China, the cultivation area is large, the demand for excellent raspberry seedlings in China is large, and many excellent varieties are introduced from foreign countries, the number is limited, and the price is high.
At present, the raspberry propagation in China mostly adopts cuttage and root-tiller seedling modes, the propagation speed is low, the multiplying power is low, the variety degradation is easy to cause, the yield is reduced, and the problem of plant diseases and insect pests is serious.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide the raspberry tissue culture seedling propagation method which is short in propagation period, high in efficiency and low in cost.
The above object of the present invention is achieved by the following technical solutions:
a tissue culture seedling propagation method of raspberries comprises the following specific steps:
s1: selecting an explant;
s2: sterilizing explants;
s3: performing induction culture;
s4: carrying out proliferation culture;
s5: rooting and culturing to obtain the root-growing culture medium,
wherein in S3, the explant disinfected in S2 is inoculated into an induction culture medium for culture, and the formula of the induction culture medium is MS + KT 0.5-1.0 mg/L + TDZ 0.005-0.01 mg/L + GA31.5-3.0 mg/L, 6g/L agar, 30g/L sucrose, 5.80 pH value of the induction culture medium,
in S4, the sterile explant in S3 is transferred to a proliferation medium, and the formula of the proliferation medium is MS + KT 0.5-1.0 mg/L + TDZ 0.002-0.015 mg/L + GA31.5-3.0 mg/L, 6g/L agar, 30g/L sucrose, pH value of the multiplication culture medium is 5.80,
in S5, the raspberry tissue culture seedling in S4 is placed in a rooting medium, the formula of the rooting medium is 1/2MS + IBA 0.5-2.0 mg/L + agar 6g/L + sucrose 15g/L, and the pH value of the rooting medium is 5.80.
By adopting the technical scheme, the buds are used as raw materials through a plant tissue culture technology, the method is convenient to obtain, the multiplication rate is up to 6-8 times, the rooting rate reaches 95%, the specifications of the seedlings are neat, the characters are kept stable, and meanwhile, the cost is saved. The germination rate of the induction culture medium can reach 90 percent, and a sterile regeneration system can be successfully established. By using the propagation culture medium, the propagation multiplying power of raspberry tissue culture seedlings can reach 6-8 times, the tissue culture seedlings are consistent in height and robust in growth, and the high multiplying power can be continuously maintained for propagating more than 15 generations. By using the rooting culture medium, the rooting rate of the raspberry tissue culture seedling can reach more than 95%, the number of roots of each raspberry is more than four, and the raspberry tissue culture seedling has a developed and robust root system.
The present invention in a preferred example may be further configured to: in S3, the culture environment after the explant disinfected in S2 is inoculated into an induction culture medium is under the condition of the illumination intensity of 1500Lux, the temperature is 25 ℃, and the illumination time is 16 h; culturing for 6 weeks under dark condition at 23 deg.C under light for 8 hr.
By adopting the technical scheme, the stress reaction of the plant material in the induction culture stage can be effectively avoided under the culture condition, the plant material can be quickly adapted to an induction culture medium, and the plant material is promoted to quickly grow and the bud is strong.
The present invention in a preferred example may be further configured to: in S4, the culture environment after inoculating the sterile explant in S3 into the proliferation medium is under the condition of the illumination intensity of 1500Lux, the temperature is 25 ℃, and the illumination time is 16 h; culturing for 8-10 weeks under the dark condition at 23 ℃ under the illumination time of 8 h.
By adopting the technical scheme, the plant material can be effectively prevented from generating stress reaction in the propagation culture stage under the culture condition, and the plant material can be quickly adapted to a propagation culture medium, so that the plant material can quickly grow, and the bud bodies are strong.
The present invention in a preferred example may be further configured to: in S4, a new growth medium is inoculated every 8 to 10 weeks.
By adopting the technical scheme, after 8-10 weeks, the effect of the proliferation culture medium is weakened, and plant materials can pollute the proliferation culture medium and need to be replaced in time.
The present invention in a preferred example may be further configured to: in S5, the culture environment of the raspberry tissue culture seedling in S4 inoculated into the rooting culture medium is under the condition of illumination intensity of 1500Lux, the temperature is 25 ℃, and the illumination time is 16 h; culturing for 4-8 weeks under the dark condition at 23 ℃ under the illumination time of 8 h.
By adopting the technical scheme, the stress reaction of the plant material in the rooting culture stage can be effectively avoided under the culture condition, the plant material can be quickly adapted to the rooting culture medium, and the plant material is promoted to quickly grow and the bud is strong.
The present invention in a preferred example may be further configured to: in S2, the specific steps of explant disinfection are that leaves of raspberry explants are cut off, the leaves are washed clean with tap water, the washed clean explants are immersed in 75% alcohol on a workbench for disinfection for 30S, the explants are washed with sterile water for 3-4 times, white cat bleaching water and sterile water are used according to the ratio of 1: 4, preparing a disinfectant, and soaking the explant in the disinfectant for 30 min.
By adopting the technical scheme, the method for disinfecting the explant is simple, the death rate of the explant can be greatly reduced, and the disinfection survival rate is high.
The present invention in a preferred example may be further configured to: in S1, the explant is selected to be the stem tip portion of a full, semi-lignified shoot of a healthy raspberry.
In summary, the invention includes at least one of the following beneficial technical effects:
according to the invention, through a plant tissue culture technology, buds are used as raw materials, the method is convenient to obtain, the multiplication rate is up to 6-8 times, the rooting rate reaches 95%, the seedling size is neat, the characters are stable, the cost is saved, the technical support is provided for industrial production of raspberries, and the method has an important significance for large-scale production of high-quality raspberries seedlings by enterprises.
Drawings
FIG. 1 is a flow chart of a tissue culture seedling propagation method of raspberry.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings.
Example (b): referring to fig. 1, the tissue culture seedling propagation method for raspberries disclosed by the invention comprises the following specific steps:
(I), test materials: the test material was taken from healthy raspberry in nursery of wuxi dongyu plant science and technology ltd, and the explant was selected from semi-lignified shoot tip with plump current-year branch buds.
(II) test method:
the raspberry tissue culture process is carried out according to the following steps: primary culture, secondary culture and rooting culture.
(1) And primary culture: cutting leaves of the raspberry explant, and washing the leaves clean with tap water. And (3) immersing the washed explants on a clean bench into 75% alcohol for disinfection for 30s, and washing with sterile water for 3-4 times. White cat bleach water and sterile water were used at a ratio of 1: 4, soaking the explant in the disinfectant for 30min, wherein the white cat bleaching water is 'white cat' brand household bleaching water produced by Shanghai white cat (group) limited company. After the disinfection is finished, the culture environment after the explant is inoculated to the induction culture medium is under the condition of illumination intensity of 1500Lux, the temperature is 25 ℃, and the illumination time is 16 h; culturing for 6 weeks at 23 deg.C under dark condition and illumination time of 8h, and establishing sterile regeneration system, wherein the induction culture medium is MS + KT 0.5-1.0 mg/L + TDZ 0.005-0.01 mg/L + GA31.5-3.0 mg/L + 6g/L agar + 30g/L sucrose, and the pH value of the induction medium is 5.80.
(2) And subculturing: taking the established sterile regeneration system as an object, and switching the sterile explant to an enrichment medium in a culture environment at the temperature of 25 ℃ for 16h under the condition of illumination intensity of 1500 Lux; culturing for 8-10 weeks in the dark at 23 ℃ under the illumination time of 8 h. Inoculating a new proliferation culture medium every 8-10 weeks, wherein the formula of the proliferation culture medium is MS + KT 0.5-1.0 mg/L + TDZ 0.002-0.015 mg/L + GA31.5-3.0 mg/L, 6g/L agar, 30g/L sucrose and 5.80 pH value of the multiplication medium.
(3) And (3) rooting culture: taking 2-3 cm terminal buds of the strong raspberry tissue culture seedlings subjected to subculture, and placing the terminal buds into a rooting culture medium in a culture environment with the illumination intensity of 1500Lux, the temperature of 25 ℃ and the illumination time of 16 h; culturing for 4-8 weeks under the dark condition at 23 ℃ under the illumination time of 8 h. Wherein the rooting culture medium comprises: 1/2MS + IBA 0.5-2.0 mg/L + agar 6g/L + sucrose 15g/L, and the pH value of the rooting medium is 5.80.
(III) test results:
primary culture: by using the disinfection method, the disinfection success rate can reach 80%, the germination rate of the induction culture medium can reach 90%, and a sterile regeneration system can be successfully established.
Subculturing: by using the proliferation culture medium disclosed by the invention, the proliferation rate of raspberry tissue culture seedlings can reach 6-8 times, the tissue culture seedlings are consistent in height and robust in growth, and the high-rate propagation can be continuously kept for more than 15 generations.
Rooting culture: by using the rooting culture medium, the rooting rate of the raspberry tissue culture seedling can reach more than 95%, the number of roots of each raspberry tissue culture seedling is more than four, and the raspberry tissue culture seedling has a developed and robust root system.
The MS culture medium of the invention is a culture medium developed by Murashige and Skoog, and the composition formula of the MS culture medium is shown in Table 1.
1/2MS culture medium contains macroelements half of MS culture medium, and other components are used in the same amount.
TABLE 1 ingredient Table of MS culture Medium
The plant growth regulator related to the invention comprises: KT-kinetin, TDZ-thidiazuron and phenylurea derivatives; GA 3-gibberellin; IBA-indolebutyric acid.
The embodiments of the present invention are preferred embodiments of the present invention, and the scope of the present invention is not limited by these embodiments, so: all equivalent changes made according to the structure, shape and principle of the invention are covered by the protection scope of the invention.
Claims (7)
1. A tissue culture seedling propagation method of raspberries is characterized in that: the method comprises the following specific steps:
s1: selecting an explant;
s2: sterilizing explants;
s3: performing induction culture;
s4: carrying out proliferation culture;
s5: rooting and culturing to obtain the root-growing culture medium,
wherein in S3, the explant disinfected in S2 is inoculated into an induction culture medium for culture, and the formula of the induction culture medium is MS + KT 0.5-1.0 mg/L + TDZ 0.005-0.01 mg/L + GA31.5-3.0 mg/L, 6g/L agar, 30g/L sucrose, 5.80 pH value of the induction culture medium,
in S4, the sterile explant in S3 is transferred to a proliferation medium, and the formula of the proliferation medium is MS + KT 0.5-1.0 mg/L + TDZ 0.002-0.015 mg/L + GA31.5-3.0 mg/L, 6g/L agar, 30g/L sucrose, pH value of the multiplication culture medium is 5.80,
in S5, the raspberry tissue culture seedling in S4 is placed in a rooting medium, the formula of the rooting medium is 1/2MS + IBA 0.5-2.0 mg/L + agar 6g/L + sucrose 15g/L, and the pH value of the rooting medium is 5.80.
2. The tissue culture seedling propagation method of raspberries as claimed in claim 1, wherein: in S3, the culture environment after the explant disinfected in S2 is inoculated into an induction culture medium is under the condition of the illumination intensity of 1500Lux, the temperature is 25 ℃, and the illumination time is 16 h; culturing for 6 weeks under dark condition at 23 deg.C under light for 8 hr.
3. The tissue culture seedling propagation method of raspberries as claimed in claim 1, wherein: in S4, the culture environment after inoculating the sterile explant in S3 into the proliferation medium is under the condition of the illumination intensity of 1500Lux, the temperature is 25 ℃, and the illumination time is 16 h; culturing for 8-10 weeks under the dark condition at 23 ℃ under the illumination time of 8 h.
4. The tissue culture seedling propagation method of raspberries as claimed in claim 3, wherein: in S4, a new growth medium is inoculated every 8 to 10 weeks.
5. The tissue culture seedling propagation method of raspberries as claimed in claim 1, wherein: in S5, the culture environment of the raspberry tissue culture seedling in S4 inoculated into the rooting culture medium is under the condition of illumination intensity of 1500Lux, the temperature is 25 ℃, and the illumination time is 16 h; culturing for 4-8 weeks under the dark condition at 23 ℃ under the illumination time of 8 h.
6. The tissue culture seedling propagation method of raspberries as claimed in claim 1, wherein: in S2, the specific steps of explant disinfection are that leaves of raspberry explants are cut off, the leaves are washed clean with tap water, the washed clean explants are immersed in 75% alcohol on a workbench for disinfection for 30S, the explants are washed with sterile water for 3-4 times, white cat bleaching water and sterile water are used according to the ratio of 1: 4, preparing a disinfectant, and soaking the explant in the disinfectant for 30 min.
7. The tissue culture seedling propagation method of raspberries as claimed in claim 1, wherein: in S1, the explant is selected to be the stem tip portion of a full, semi-lignified shoot of a healthy raspberry.
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| CN201911161594.3A CN110810241A (en) | 2019-11-22 | 2019-11-22 | Tissue culture seedling propagation method for raspberries |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115152629A (en) * | 2022-07-26 | 2022-10-11 | 辽宁省果树科学研究所 | Raspberry tissue culture method |
| CN115968778A (en) * | 2022-10-19 | 2023-04-18 | 北京大学现代农业研究院 | Method for in vitro regeneration culture of raspberries |
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| WO2018096212A1 (en) * | 2016-11-24 | 2018-05-31 | Teknologian Tutkimuskeskus Vtt Oy | Arctic bramble (rubus arcticus) cell cultures, method for producing arctic bramble cell cultures, compositions comprising arctic bramble cell cultures and use of arctic bramble cell cultures |
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- 2019-11-22 CN CN201911161594.3A patent/CN110810241A/en active Pending
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115152629A (en) * | 2022-07-26 | 2022-10-11 | 辽宁省果树科学研究所 | Raspberry tissue culture method |
| CN115968778A (en) * | 2022-10-19 | 2023-04-18 | 北京大学现代农业研究院 | Method for in vitro regeneration culture of raspberries |
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