CN113331058A - Damascus rose tissue culture method - Google Patents
Damascus rose tissue culture method Download PDFInfo
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- CN113331058A CN113331058A CN202110798722.6A CN202110798722A CN113331058A CN 113331058 A CN113331058 A CN 113331058A CN 202110798722 A CN202110798722 A CN 202110798722A CN 113331058 A CN113331058 A CN 113331058A
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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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- 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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- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
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Abstract
The invention relates to the technical field of plant tissue culture, in particular to a culture medium and a culture method for tissue culture and propagation of rosa damascena. The tissue culture propagation culture medium comprises an induction culture medium, a proliferation culture medium and a rooting culture medium; the induction culture medium, the proliferation culture medium and the rooting culture medium all use a Huabao (HYPONEX) culture medium as a basic culture medium. The invention breakthroughs in the use of Huabao fertilizer to replace major elements such as nitrate in MS basic culture medium, establishes a set of complete Damascus rose tissue culture system, and the cultured plants are strong and have dark green leaf color, the multiplication coefficient reaches more than 2.5, the rooting rate reaches 80%, and the transplanting survival rate reaches 95%.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a culture medium and a culture method for tissue culture and propagation of rosa damascena.
Background
The rose has bright color, pleasant fragrance and high oil content, is an important special economic plant in China, is a national secondary protective plant, and is one of important ornamental and production dual-purpose flowers in the world. It can be enjoyed by people, and is also an important raw material for precious traditional Chinese medicinal materials, food industry, spice industry and the like. Fresh rose contains various trace elements and has quite high content of vitamin C, and natural essence and spice products extracted from roses have extremely high economic value and are one of important raw materials for preparing high-grade essence, cigarettes, printing ink, high-grade perfume, high-grade cosmetics and the like.
At present, the basic technology of plant tissue culture is mature, the period is short, the culture condition is convenient to manually control, the propagation speed is high, the occupied space is small, the economic benefit is high, and the method is an effective way for cultivating excellent varieties. The production rate, survival quality and ornamental quality of the rose flowers are greatly improved by utilizing the plant rapid propagation technology.
The existing propagation method of the rosa damascena is mainly cutting propagation, the survival rate is low, the rooting time is long, and the quality of offspring is reduced. At present, the plant tissue culture method is used for propagation, but the survival rate of transplanting tissue culture seedlings is low, so that the industrialized production of the rosa damascena is greatly limited
Based on the situation, the invention provides a culture medium and a culture method for tissue culture and propagation of rosa damascena, which can effectively solve the problems.
Disclosure of Invention
The invention aims to provide a culture medium and a culture method for tissue culture and propagation of rosa damascena.
In order to realize the aim, the invention provides a culture medium for tissue culture and propagation of rosa damascena, which comprises an induction culture medium, a multiplication culture medium and a rooting culture medium; the induction culture medium, the proliferation culture medium and the rooting culture medium all use culture media of components except major elements of Huabao (HYPONEX) and MS as basal culture media.
Preferably, the induction culture medium takes ingredients except the macroelements of Huabao No. 1 and MS as a basic culture medium.
Preferably, the proliferation culture medium takes ingredients except the major elements of Huabao No. 1 and MS as a basic culture medium.
Preferably, the rooting culture medium takes ingredients except the major elements of Huabao No. 1 and MS as a basic culture medium.
Preferably, the induction culture medium further comprises 1.0-2.0 mg/L of 6-benzylaminopurine, 0.05-0.2 mg/L of naphthylacetic acid, and the pH value is 5.6-6.0.
Preferably, the multiplication medium further comprises 1.0-2.0 mg/L of 6-benzylaminopurine, and the pH value is 5.6-6.0.
Preferably, the rooting medium further comprises 0.1-0.5 mg/L of naphthylacetic acid, 0.2-0.8 mg/L of indoleacetic acid, and the pH value is 5.6-6.0.
The invention also provides a culture method for tissue culture propagation of rosa damascena, which comprises the following steps:
taking stems of the rosa damascena as explants, carrying out induction culture in an induction culture medium to generate inorganic salt, carrying out subculture on the germplasm tissues in the subculture medium to generate sterile buds, carrying out multiplication culture on the sterile buds in the multiplication culture medium to generate cluster buds, and carrying out rooting culture on the cluster buds in the rooting culture medium to form rosa damascena seedlings.
Preferably, the method comprises the steps of:
(1) preparing various culture media according to the proportion of the various culture media;
(2) collecting the current-year-old twigs of Damascus as explants, stripping off hard spines on the twigs, wiping the surfaces of the twigs clean with alcohol, and then cutting the twigs into 2-3 cm-long stem sections, wherein each section has one node. Soaking the stem sections in 75% ethanol for 30-40 s, washing with sterile water for 1-2 times, soaking with 0.1% mercuric chloride for 10-15 min, shaking the container continuously during the soaking, and washing with sterile water for 5-6 times after treatment;
(3) inoculating the stem segments into an induction culture medium, culturing in a light-tight environment at 21 +/-2 ℃ for 5-7 days, transferring into a culture room at 23 +/-2 ℃ for illumination for 10-12 h/day, wherein the illumination intensity is 1600-2000 Lx, and the culture time is 1 month, so as to obtain sterile buds of the rosa damascena;
(4) transferring the sterile buds of the rosa damascena in the step (3) into a multiplication culture medium, and subculturing once every 20-35 days under the conditions that the temperature is 23 +/-2 ℃, the illumination intensity is 1600-2000 Lx and the illumination is 12-14 h every day to obtain rosette buds;
(5) cutting the cluster buds of the rosa damascena obtained in the step (4) into single plants, transferring the single plants into a rooting culture medium, and culturing for 24-30 days at the temperature of 25 +/-2 ℃, the illumination intensity of 2500-3000 Lx and illumination time of 12-16 h every day to obtain the rosa damascena seedlings.
The Huabao fertilizers adopted by the invention are American HYPONEX Huabao No. 1. Wherein, the contents of nitrogen, phosphorus and potassium of Huabao No. 1 are respectively 7: 6: 9.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention breakthroughs in the use of Huabao fertilizer to replace major elements such as nitrate in MS basic culture medium, establishes a set of complete Damascus rose tissue culture system, and the cultured plants are strong and have dark green leaf color, the multiplication coefficient reaches more than 2.5, the rooting rate reaches 80%, and the transplanting survival rate reaches 95%.
2. The raw materials of the invention are sufficient in China and proper in price, so that the large-scale production of the invention is not limited by too high cost; meanwhile, the preparation method is simple, the total production cost is low, and the industrial large-scale production is facilitated.
Detailed Description
Example 1
(1) Weighing specific raw materials according to table 1, and preparing various culture media;
(2) collecting the current-year-old twigs of Damascus as explants, stripping off hard spines on the twigs, wiping the surfaces of the twigs clean with alcohol, and then cutting the twigs into 2-3 cm-long stem sections, wherein each section has one node. Soaking the stem sections in 75% ethanol for 30-40 s, washing with sterile water for 1-2 times, soaking with 0.1% mercuric chloride for 10-15 min, shaking the container continuously during the soaking, and washing with sterile water for 5-6 times after treatment;
(3) inoculating the stem segments into an induction culture medium, culturing in a light-tight environment at 21 +/-2 ℃ for 5-7 days, transferring into a culture room at 23 +/-2 ℃ for illumination for 10-12 h/day, wherein the illumination intensity is 1600-2000 Lx, and the culture time is 1 month, so as to obtain sterile buds of the rosa damascena;
(4) cutting the sterile bud of the rosa damascena in the step (3) into independent buds, transferring the independent buds into a proliferation culture medium, and subculturing once every 20 days and subculturing 2 times under the conditions that the temperature is 23 +/-2 ℃, the illumination intensity is 1600Lx and the illumination is 12 hours every day to obtain the rosette cluster buds;
(5) cutting the cluster buds of the rosa damascena obtained in the step (4) into single plants, transferring the single plants into a rooting culture medium, and culturing for 30 days at the temperature of 25 +/-2 ℃, the illumination intensity of 2500Lx and the illumination time of 13h every day to obtain the rosa damascena seedlings.
Example 2
(1) Weighing specific raw materials according to table 1, and preparing various culture media;
(2) collecting the current-year-old twigs of Damascus as explants, stripping off hard spines on the twigs, wiping the surfaces of the twigs clean with alcohol, and then cutting the twigs into 2-3 cm-long stem sections, wherein each section has one node. Soaking the stem sections in 75% ethanol for 30-40 s, washing with sterile water for 1-2 times, soaking with 0.1% mercuric chloride for 10-15 min, shaking the container continuously during the soaking, and washing with sterile water for 5-6 times after treatment;
(3) inoculating the stem segments into an induction culture medium, culturing in a light-tight environment at 21 +/-2 ℃ for 5-7 days, transferring into a culture room at 23 +/-2 ℃ for illumination for 10-12 h/day, wherein the illumination intensity is 1600-2000 Lx, and the culture time is 1 month, so as to obtain sterile buds of the rosa damascena;
(4) cutting the sterile bud of the rosa damascena in the step (3) into independent buds, transferring the independent buds into a proliferation culture medium, and subculturing for 2 times every 25 days under the conditions that the temperature is 23 +/-2 ℃, the illumination intensity is 2000Lx and the illumination is 14 hours every day to obtain the rosette cluster buds;
(5) cutting the cluster buds of the rosa damascena obtained in the step (4) into single plants, transferring the single plants into a rooting culture medium, and culturing for 24 days at the temperature of 25 +/-2 ℃, the illumination intensity of 3000Lx and the illumination time of 16h every day to obtain the rosa damascena seedlings.
Example 3
(1) Weighing specific raw materials according to table 1, and preparing various culture media;
(2) collecting the current-year-old twigs of Damascus as explants, stripping off hard spines on the twigs, wiping the surfaces of the twigs clean with alcohol, and then cutting the twigs into 2-3 cm-long stem sections, wherein each section has one node. Soaking the stem sections in 75% ethanol for 30-40 s, washing with sterile water for 1-2 times, soaking with 0.1% mercuric chloride for 10-15 min, shaking the container continuously during the soaking, and washing with sterile water for 5-6 times after treatment;
(3) inoculating the stem segments into an induction culture medium, culturing in a light-tight environment at 21 +/-2 ℃ for 5-7 days, transferring into a culture room at 23 +/-2 ℃ for illumination for 10-12 h/day, wherein the illumination intensity is 1600-2000 Lx, and the culture time is 1 month, so as to obtain sterile buds of the rosa damascena;
(4) dividing adventitious buds of the Damascus rose in the step (3) into independent buds, transferring the independent buds into a proliferation culture medium, and subculturing for 2 times every 20 days under the conditions that the temperature is 23 +/-2 ℃, the illumination intensity is 1600Lx and the illumination is 12 hours every day to obtain cluster buds of the Damascus rose;
(5) cutting the cluster buds of the rosa damascena obtained in the step (4) into single plants, transferring the single plants into a rooting culture medium, and culturing for 30 days at the temperature of 25 +/-2 ℃, the illumination intensity of 2500Lx and the illumination time of 13h every day to obtain the rosa damascena seedlings.
TABLE 1
Example 4 culture Effect test
Taking the embodiments 1-3 of the invention as objects, counting the survival rate of adventitious buds of Rosa damascena after culturing in a subculture medium; carrying out enrichment culture in an enrichment culture medium and then counting the multiplication coefficient of rosette buds; counting the rooting rate of the rosa damascena seedlings after rooting culture in a rooting culture medium; and then, transferring the rosa damascena seedlings outdoors, controlling the temperature to be 20-30 ℃ and the relative air humidity to be 75-85% after transplanting, normally managing water, fertilizer and pesticide, and counting the transplanting survival rate after transplanting for 35 d.
The test results are shown in Table 2.
TABLE 2 culture Effect test
The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.
Claims (8)
1. A culture medium for tissue culture and propagation of Rosa damascena is characterized by comprising an induction culture medium, a proliferation culture medium and a rooting culture medium; the induction culture medium, the proliferation culture medium and the rooting culture medium all use a Huabao (HYPONEX) culture medium as a basic culture medium.
2. The tissue culture propagation culture medium of claim 1, wherein the induction culture medium is a basal culture medium containing components except macroelements of Huabao No. 1 and MS.
3. The tissue culture propagation medium of claim 1, wherein the propagation medium is a basal medium containing components except macroelements of Huabao No. 1 and MS.
4. The tissue culture propagation culture medium of claim 1, wherein the rooting culture medium is a basal culture medium containing components except macroelements of Huabao No. 1 and MS.
5. The tissue culture propagation medium of claim 2, wherein the induction medium further comprises 1.0-2.0 mg/L of 6-benzylaminopurine, 0.05-0.2 mg/L of naphthylacetic acid, and the pH value is 5.6-6.0.
6. The tissue culture propagation medium according to claim 3, wherein the propagation medium further comprises 1.0-2.0 mg/L of 6-benzylaminopurine, 0.1-0.3 mg/L of indoleacetic acid, and the pH value is 5.6-6.0.
7. The tissue culture propagation medium according to claim 4, wherein the rooting medium further comprises 0.1-0.5 mg/L of naphthylacetic acid, 0.2-0.8 mg/L of indolylacetic acid, and the pH value is 5.6-6.0.
8. A culture method for tissue culture propagation of rosa damascena is characterized by comprising the following steps: taking stems of the rosa damascena as explants, carrying out induction culture in an induction culture medium to generate inorganic salt, carrying out proliferation culture on inorganic buds in a proliferation culture medium to generate cluster buds, and carrying out rooting culture on the cluster buds in a rooting culture medium to form rosa damascena seedlings.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103960133A (en) * | 2014-05-27 | 2014-08-06 | 昆明学院 | Method for tissue culture and rapid propagation of Rosa rugosa Thunb. |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103960133A (en) * | 2014-05-27 | 2014-08-06 | 昆明学院 | Method for tissue culture and rapid propagation of Rosa rugosa Thunb. |
Non-Patent Citations (4)
| Title |
|---|
| 崔兴林: "《苦水玫瑰栽培与加工技术》", 31 January 2019, 天津大学出版社 * |
| 殷建宝: "《植物组织培养快繁技术》", 30 November 2018, 阳光出版社 * |
| 熊丽: "《观赏花卉的组织培养与大规模生产》", 31 January 2003, 化学工业出版社 * |
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